Methods and compositions useful for inhibition of angiogenesis

ABSTRACT

The present invention describes methods for inhibition of angiogenesis in tissues using vitronectin α v β 3  antagonists, and particularly for inhibiting angiogenesis in inflamed tissues and in tumor tissues and metastases using therapeutic compositions containing α v β 3  antagonists.

This application is a 371 of PCT US97/09158, this application claims thebenefit of provisional application 60/018773 filed May 31, 1996 and60/015869 filed May 31, 1996.

This invention was made with government support under Contract Nos.CA50826, CA45726, HL54444, T32 AI07244-11 and F32 CA72192 by theNational Institutes of Health. The government has certain rights in theinvention.

TECHNICAL FIELD

The present invention relates generally to the field of medicine, andrelates specifically to methods and compositions for inhibitingangiogenesis of tissues using antagonists of the vitronectin receptorα_(v)β₃.

BACKGROUND

Integrins are a class of cellular receptors known to bind extracellularmatrix proteins, and therefore mediate cell-cell and cell-extracellularmatrix interactions, referred generally to as cell adhesion events.However, although many integrins and the ligands that bind an integrinare described in the literature, the biological function of many of theintegrins remains elusive. The integrin receptors constitute a family ofproteins with shared structural characteristics of noncovalentheterodimeric glycoprotein complexes formed of α and β subunits.

The vitronectin receptor, named for its original characteristic ofpreferential binding to vitronectin, is now known to refer to threedifferent integrins, designated α_(v)β₁, α_(v)β₃ and α_(v)β₅. Horton,Int. J. Exp. Pathol., 71:741-759 (1990). α_(v)β₁ binds fibronectin andvitronectin. α_(v)β₃ binds a large variety of ligands, including fibrin,fibrinogen, laminin, thrombospondin, vitronectin, von Willebrand'sfactor, osteospontin and bone sialoprotein I. α_(v)β₅ binds vitronectin.The specific cell adhesion roles these three integrins play in the manycellular interactions in tissues are still under investigation, but itis clear that there are different integrins with different biologicalfunctions.

One important recognition site in the ligand for many integrins is thearginine-glycine-aspartic acid (RGD) tripeptide sequence. RGD is foundin all of the ligands identified above for the vitronectin receptorintegrins. This RGD recognition site can be mimicked by polypeptides(“peptides”) that contain the RGD sequence, and such RGD peptides areknown inhibitors of integrin function. It is important to note, however,that depending upon the sequence and structure of the RGD peptide, thespecificity of the inhibition can be altered to target specificintegrins.

For discussions of the RGD recognition site, see Pierschbacher et al.,Nature, 309:30-33 (1984), and Pierschbacher et al., Proc. Natl. Acad.Sci., USA, 81:5985-5988 (1984). Various RGD polypeptides of varyingintegrin specificity have also been described by Grant et al., Cell,58:933-943 (1989), Cheresh et al., Cell, 58:945-953 (1989), Aumailley etal., FEBS Letts., 291:50-54 (1991), and Pfaff et al., J. Biol. Chem.,269:20233-20238 (1994), and in U.S. Pat. Nos. 4,517,686, 4,578,079,4,589,881, 4,614,517, 4,661,111, 4,792,525, 4,683,291, 4,879,237,4,988,621, 5,041,380 and 5,061,693.

Angiogenesis is a process of tissue vascularization that involves thegrowth of new developing blood vessels into a tissue, and is alsoreferred to as neo-vascularization. The process is mediated by theinfiltration of endothelial cells and smooth muscle cells. The processis believed to proceed in any one of three ways: the vessels can sproutfrom pre-existing vessels, de-novo development of vessels can arise fromprecursor cells (vasculogenesis), or existing small vessels can enlargein diameter. Blood et al., Bioch. Biophys. Acta, 1032:89-118 (1990).Vascular endothelial cells are known to contain at least fiveRGD-dependent integrins, including the vitronectin receptor (α_(v)β₃ orα_(v)β₅), the collagen Types I and IV receptor (α₁β₁), the lamininreceptor (α₂β₁), the fibronectin/laminin/collagen receptor (α₁β₁) andthe fibronectin receptor (α₅β₁ ). Davis et al., J. Cell. Biochem.,51:206-218 (1993). The smooth muscle cell is known to contain at leastsix RGD-dependent integrins, including α₅β₁, α_(v)β₃ and α_(v)β₅.

Angiogenesis is an important process in neonatal growth, but is alsoimportant in wound healing and in the pathogenesis of a large variety ofclinical diseases including tissue inflammation, arthritis, tumorgrowth, diabetic retinopathy, macular degeneration by neovascularizationof retina and the like conditions. These clinical manifestationsassociated with angiogenesis are referred to as angiogenic diseases.Folkman et al., Science, 235:442-447 (1987). Angiogenesis is generallyabsent in adult or mature tissues, although it does occur in woundhealing and in the corpeus leuteum growth cycle. See, for example, Moseset al., Science, 248:1408-1410 (1990).

It has been proposed that inhibition of angiogenesis would be a usefultherapy for restricting tumor growth. Inhibition of angiogenesis hasbeen proposed by (1) inhibition of release of “angiogenic molecules”such as bFGF (basic fibroblast growth factor), (2) neutralization ofangiogenic molecules, such as by use of anti-βbFGF antibodies, and (3)inhibition of endothelial cell response to angiogenic stimuli. Thislatter strategy has received attention, and Folkman et al., CancerBiology, 3:89-96 (1992), have described several endothelial cellresponse inhibitors, including collagenase inhibitor, basement membraneturnover inhibitors, angiostatic steroids, fungal-derived angiogenesisinhibitors, platelet factor 4, thrombospondin, arthritis drugs such asD-penicillamine and gold thiomalate, vitamin D₃ analogs,alpha-interferon, and the like that might be used to inhibitangiogenesis. For additional proposed inhibitors of angiogenesis, seeBlood et al., Bioch. Biophys. Acta., 1032:89-118 (1990), Moses et al.,Science, 248:1408-1410 (1990), Ingber et al., Lab. Invest., 59:44-51(1988), and U.S. Pat. Nos. 5,092,885, 5,112,946, 5,192,744, and5,202,352. None of the inhibitors of angiogenesis described in theforegoing references are targeted at inhibition of α_(v)β₃.

RGD-containing peptides that inhibit vitronectin receptor α_(v)β₃ havealso been described. Aumailley et al., FEBS Letts., 291:50-54 (1991),Choi et al., J. Vasc. Surg., 19:125-134 (1994), Smith et al., J. Biol.Chem., 265:12267-12271 (1990), and Pfaff et al., J. Biol. Chem.,269:20233-20238 (1994). However, the role of the integrin α_(v)β₃ inangiogenesis has never been suggested nor identified until the presentinvention.

For example, Hammes et al., Nature Med., 2:529-53 (1996) confirmed thefindings of the present invention. Specifically, the paper shows thatcyclic peptides including cyclic RGDfV, the structure and function ofthe latter of which has been previously described in the priorityapplications on which the present application is based, inhibitedretinal neovascularization in a mouse model of hypoxia-induced retinalneovascularization. In a separate study that also supports the presentinvention as well as the priority applications, Luna et al., Lab.Invest., 75:563-573 (1996) described two particular cyclic methylatedRGD-containing peptides that were partially effective at inhibitingretinal neovascularization in the mouse model of oxygen-induced ischemicretinopathy. In contrast, the peptides of the present invention exhibitalmost complete inhibition of neovascularization in the model systemsdescribed herein.

Inhibition of cell adhesion in vitro using monoclonal antibodiesimmunospecific for various integrin α or β subunits have implicatedα_(v)β₃ in cell adhesion of a variety of cell types includingmicrovascular endothelial cells. Davis et al., J. Cell. Biol.,51:206-218 (1993). In addition, Nicosia et al., Am. J. Pathol.,138:829-833 (1991), described the use of the RGD peptide GRGDS to invitro inhibit the formation of “microvessels” from rat aorta cultured incollagen gel. However, the inhibition of formation of “microvessels” invitro in collagen gel cultures is not a model for inhibition ofangiogenesis in a tissue because it is not shown that the microvesselstructures are the same as capillary sprouts or that the formation ofthe microvessel in collagen gel culture is the same as neovasculargrowth into an intact tissue, such as arthritic tissue, tumor tissue ordisease tissue where inhibition of angiogenesis is desirable.

For angiogenesis to occur, endothelial cells must first degrade andcross the blood vessel basement membrane in a similar manner used bytumor cells during invasion and metastasis formation.

The inventors have previously reported that angiogenesis depends on theinteraction between vascular integrins and extracellular matrixproteins. Brooks et al., Science, 264:569-571 (1994). Furthermore, itwas reported that programmed cell death (apoptosis) of angiogenicvascular cells is initiated by the interaction, which would beinhibitied by certain antagonists of the vascular integrin α_(v)β₃.Brooks et al., Cell, 79:1157-1164 (1994). More recently, the inventorshave reported that the binding of matrix metalloproteinase-2 (MMP-2) tovitronectin receptor ( can be inhibited using α_(v)β₅ antagonists, andthereby inhibit the enzymatic function of the proteinase. Brooks et al.,Cell, 85:683-693 (1996).

Other than the studies reported here, Applicants are unaware of anyother demonstration that angiogenesis could be inhibited in a tissueusing inhibitors of cell adhesion. In particular, it has never beenpreviously demonstrated by others that α_(v)β₃ function is required forangiogenesis in a tissue or that α_(v)β₃ antagonists can inhibitangiogenesis in a tissue.

BRIEF DESCRIPTION OF THE INVENTION

The present invention disclosure demonstrates that angiogenesis intissues requires integrin α_(v)β₃, and that inhibitors of α_(v)β₃ caninhibit angiogenesis. The disclosure also demonstrates that antagonistsof other integrins, such as α_(IIb)β₃, or α_(v)β₁, do not inhibitangiogenesis, presumably because these other integrins are not essentialfor angiogenesis to occur.

The invention therefore describes methods for inhibiting angiogenesis ina tissue comprising administering to the tissue a composition comprisingan angiogenesis-inhibiting amount of an α_(v)β₃ antagonist.

The tissue to be treated can be any tissue in which inhibition ofangiogenesis is desirable, such as diseased tissue whereneo-vascularization is occurring. Exemplary tissues include inflamedtissue, solid tumors, metastases, tissues undergoing restenosis, and thelike tissues.

An α_(v)β₃ antagonist for use in the present methods is capable ofbinding to α_(v)β₃ and competitively inhibiting the ability of α_(v)β₃to bind to a natural ligand. Preferably, the antagonist exhibitsspecificity for α_(v)β₃ over other integrins. In a particularlypreferred embodiment, the α_(v)β₃ antagonist inhibits binding offibrinogen or other RGD-containing ligands to α_(v)β₃ but does notsubstantially inhibit binding of fibrinogen to α_(IIb)β₃. A preferredα_(v)β₃ antagonist can be a fusion polypeptied, a cyclic or linearpolypeptide, a derivatized polypeptide, a monoclonal antibody thatimmunoreacts with α_(v)β₃, an organic mimetic of α_(v)β₃ or functionalfragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings forming a portion of this disclosure:

FIGS. 1A-1D illustrate the tissue distribution of the integrin subunits,β₃ and β₁, in normal skin and in skin undergoing wound healingdesignated as granulation tissue. Immunohistochemistry with antibodiesto β₃ and β₁ was performed as described in Example 3A. FIGS. 1A and 1Brespectively illustrate the immunoreactivity of anti-β₃ in normal skinand granulation tissue. FIGS. 1C and 1D respectively illustrate theimmunoreactivity of anti-β₁ in normal skin and granulation tissue.

FIGS. 2A-2D illustrate the tissue distribution of the von Willebrandfactor and laminin ligands that respectively bind the β₃ and β₁ integrinsubunits in normal skin and in skin undergoing wound healing designatedas granulation tissue. Immunohistochemistry with antibodies to vonWillebrand factor (anti-vWF) and laminin (anti-laminin) was performed asdescribed in Example 3B. FIGS. 2A and 2B respectively illustrate theimmunoreactivity of anti-vWF in normal skin and granulation tissue.FIGS. 2C and 2D respectively illustrate the immunoreactivity ofanti-laminin in normal skin and granulation tissue.

FIGS. 3A-3D illustrate the tissue distribution of the vitronectinintegrin receptor, α_(v)β₃, in tissue biopsies of bladder cancer, coloncancer, breast cancer and lung cancer, respectively.Immunohistochemistry with the LM609 antibody against α_(v)β₃ wasperformed as described in Example 3C.

FIG. 4 illustrates a typical photomicrograph of a CAM of this inventiondevoid of blood vessels in an untreated 10 day old chick embryo. Thepreparation is described in Example 5B.

FIGS. 5A-5C illustrate the tissue distribution of the integrins β₁ andα_(v)β₃ in the CAM preparation of this invention. FIG. 5A shows thedistribution of the β₁ subunit in an untreated 10 day old CAMpreparation as detected by immunofluorescence immunoreactivity withCSAT, an anti-β₁ antibody. FIG. 5B shows the distribution of the α_(v)β₃receptor in an untreated 10 day old CAM preparation as detected byimmunofluorescence immunoreactivity with LM609, an anti-α_(v)β₃antibody. FIG. 5C shows the distribution of the α_(v)β₃ receptor in anbFGF treated 10 day old CAM preparation as detected byimmunofluorescence immunoreactivity with LM609, an anti-α_(v)β₃antibody. The treatments and results are described in Example 5C.

FIG. 6 illustrates the quantification in a bar graph of the relativeexpression of α_(v)β₃ and β₁ in untreated and bFGF treated 10 day oldCAMs as described in Example 6A. The mean fluorescence intensity isplotted on the Y-axis with the integrin profiles plotted on the X-axis.

FIGS. 7A-7C illustrates the appearance of an untreated 10 day old CAM, abFGF treated CAM, and a TNFα treated CAM, respectively, the proceduresand results of which are described in Example 6A.

FIGS. 8A-8E illustrate the effect of topical antibody treatment onbFGF-induced angiogenesis in a day 10 CAM as described in Example 7A1).FIG. 8A shows an untreated CAM preparation that is devoid of bloodvessels, FIG. 8B shows the infiltration of new vasculature into an areapreviously devoid of vasculature induced by bFGF treatment. FIGS. 8C, 8Dand 8E respectively show the effects of antibodies against β₁ (anti-β₁;CSAT) , α_(v)β₅ (anti-α_(v)β₅; P3G2) and α_(v)β₃ (anti-α_(v)β₃; LM609).

FIGS. 9A-9C illustrate the effect of intravenous injection of syntheticpeptide 66203 on angiogenesis induced by tumors as described in Example7E2). FIG. 9A shows the lack of inhibitory effect of intravenoustreatment with a control peptide (control peptide tumor) on angiogenesisresulting from tumor induction. The inhibition of such angiogenesis byintravenous injection of peptide 66203 (cyclic RGD tumor) is shown inFIG. 9B. The lack of inhibitory effects or cytotoxicity on maturepreexisting vessels following intravenous infusion of peptide 66203 inan area adjacent to the tumor-treated area is shown in FIG. 9C (cyclicRGD adjacent CAM).

FIGS. 10A-10C illustrate the effect of intravenous application ofmonoclonal antibodies to growth factor induced angiogenesis as describedin Example 7B1). FIG. 10A shows bFGF-induced angiogenesis not exposed toantibody treatment (control). No inhibition of angiogenesis resultedwhen a similar preparation was treated with anti-α_(v)β₅ antibody P3G2as shown in FIG. 10B. Inhibition of angiogenesis resulted with treatmentof anti-α_(v)β₃ antibody LM609 as shown in FIG. 10C.

FIGS. 11A-11C illustrate the effect on embryonic angiogenesis followingtopical application of anti-integrin antibodies as described in Example7C. Angiogenesis was not inhibited by treatment of a 6 day CAM withanti-β₁ and anti-α_(v)β₅ antibodies respectively shown in FIGS. 11A and11B. In contrast, treatment with the anti-α_(v)β₃ antibody LM609resulted in the inhibition of blood vessel formation as shown in FIG.11C.

FIG. 12 illustrates the quantification of the number of vessels enteringa tumor in a CAM preparation as described in Example 7D1). The graphshows the number of vessels as plotted on the Y-axis resulting fromtopical application of either CSAT (anti-α₁) , LM609 (anti-α_(v)β₃) orP3G2 (anti-α_(v)β₃).

FIGS. 13A-13D illustrate a comparison between wet tumor weights 7 daysfollowing treatment and initial tumor weights as described in Example9A1)a. Each bar represents the mean ±S.E. of 5-10 tumors per group.Tumors were derived from human melanoma (M21-L) (FIG. 13A), pancreaticcarcinoma (Fg) (FIG. 13B), lung carcinoma (UCLAP-3) (FIG. 13C), andlaryngeal carcinoma (HEp3) (FIG. 13D) CAM preparations and treatedintravenously with PBS, CSAT (anti-β₁), or LM609 (anti-α_(v)β₃). Thegraphs show the tumor weight as plotted on the Y-axis resulting fromintravenous application of either CSAT (anti-β₁), LM609 (anti-α_(v)β₃)or PBS as indicated on the X-axis.

FIGS. 14A and 14B illustrate histological sections of tumors treatedwith the P3G2 (anti-α_(v)β₅) (FIG. 14A) and LM609 (anti-α_(v)β₃) (FIG.14B), and stained with hematoxylin and eosin as described in Example9A1)b. As shown in FIG. 14A, tumors treated with control antibody (P3G2)showed numerous viable and actively dividing tumor cells as indicated bymitotic figures (arrowheads) as well as by multiple blood vessels(arrows) throughout the tumor stroma. In contrast, few if any viabletumor cells or blood vessels were detected in tumors treated with LM609(anti-α_(v)β₃) in FIG. 14B.

FIGS. 15A-15E correspond to M21L tumors treated with peptides asdescribed in Example 9A2) and are as follows: FIG. 15A, control cyclicRAD peptide (69601); FIG. 15B, cyclic RGD peptide (66203); FIG. 15C,adjacent CAM tissue taken from the same embryos treated with cyclic RGDpeptide (66203) and high magnification (13×) of tumors treated with thecontrol RAD (69601) in FIG. 15D or cyclic RGD peptide (66203) in FIG.15E. FIG. 15D depicts normal vessels from the RAD control peptide(69601) treated tumor. FIG. 15E depicts examples of disrupted bloodvessels from cyclic RGD peptide (66203) treated tumors (arrows).

FIGS. 16A-16E represent inhibition of angiogenesis by antagonists ofangiogenesis in the in vivo rabbit eye model assay as described inExample 10. FIGS. 16A and 16B depict angiogenesis of the rabbit eye inthe presence of bFGF and mAb P1F6 (anti-α_(v)β₅). FIGS. 16C, 16D and 16Edepict inhibition of angiogenesis of the rabbit eye in the presence ofbFGF and mAb LM609 (anti-α_(v)β₃).

FIG. 17 represents a flow chart of how the in vivo mouse:human chimericmouse model was generated as described in Example 11. A portion of skinfrom a SCID mouse was replaced with human neonatal foreskin and allowedto heal for 4 weeks. After the graft had healed, the human foreskin wasinoculated with human tumor cells. During the following 4 week period, ameasurable tumor was established which comprised a human tumor withhuman vasculature growing from the human skin into the human tumor.

FIG. 18 illustrates the percent of single cells derived from mAb-treatedand peptide-treated CAMs and stained with Apop Tag as determined by FACSanalysis and described in Example 12. The black and stippled barsrepresent cells from embryos treated 24 hours and 48 hours prior to theassay, respectively. Each bar represents the mean±S.E. of threereplicates. CAMs were treated mAb LM609 (anti-α_(v)β₃), or CSAT(anti-α₁), or PBS. CAMs were also treated with cyclic peptide 66203(cyclo-RGDfV, indicated as Peptide 203) or control cyclic peptide 69601(cyclo-RADfV, indicated as Peptide 601).

FIGS. 19A and 19B illustrate the combined results of single cellsuspensions of CAMs from embryos treated with either CSAT (anti-β₁)(FIG. 19A) or LM609 (ant-α_(v)β₃) (FIG. 19B), stained with Apop Tag andpropidium iodide, and analyzed by FACS as described in Example 12C. TheY axis represents Apop Tag staining in numbers of cells (Apoptosis), theX axis represents propidium iodide staining (DNA content). Thehorizontal line represents the negative gate for Apop Tag staining. Theleft and right panels indicates CAM cells from CSAT (anti-β₁) (FIG. 19A)and LM609 (anti-α_(v)β₃) (FIG. 19B) treated embryos, respectively. Cellcycle analysis was performed by analysis of approximately 8,000 eventsper condition.

FIGS. 20A-20C represent CAM tissue from CSAT (anti-β₁) treated embryosand FIGS. 20D-20F represent CAM tissue from LM609 (anti-α_(v)β₃) treatedembryos prepared as described in Example 12C. FIGS. 20A and 20D depicttissues stained with Apop Tag and visualized by fluorescence (FITC)superimposed on a D.I.C. image. FIGS. 20B and 20E depict the sametissues stained with mAb LM609 (anti-α_(v)β₃) and visualized byfluorescence (rhodamine). FIGS. 20C and 20F represent merged images ofthe same tissues stained with both Apop Tag and LM609 where yellowstaining represents colocalization. The bar represents 15 and 50 μm inthe left and right panels, respectively.

FIG. 21 shows the result of a inhibition of cell attachment assay withpeptide 85189 as described in Example 4A. The effects of the peptideantagonist was assessed over a dosage range of 0.001 to 100 uM asplotted on the X-axis. Cell attachment is plotted on the Y-axis measuredat an optical density (O.D.) of 600 nm. Cell attachment was measured onvitronectin- (broken lines) versus laminin- (solid lines) coatedsurfaces.

FIGS. 22A and 22B show the consecutive cDNA sequence of chicken MMP-2along with the deduced amino acid sequence shown on the second line. Thethird and fourth lines respectively show the deduced amino acid sequenceof human and mouse MMP-2 as described in Example 4A. The chicken cDNAsequence is listed in SEQ ID NO 29 along with the encoded amino acidsequence that is also presented separately as SEQ ID NO 30. Thenumbering of the first nucleotide of the 5′ untranslated region and theregion encoding the proenzyme sequence shown in FIG. 22A as a negativenumber is actually presented as number 1 in Sequence Listing making thelatter appear longer than the figure; however, the nucleotide sequenceis the figure is identical in length and sequence to that as presentedin the listing with the exception of the numbering. Accordingly,references to nucleotide position for chicken or human MMP-2 in thespecification, such as in primers for use in amplifying MMP-2 fragments,are based on the nucleotide position as indicated in the figure and notas listed in the Sequence Listing.

FIG. 23 shows the results in bar-graph form of a solid-phase receptorbinding assay of iodinated MMP-2 to bind to α_(v)β₃ with and without thepresence of inhibitors as further described in Example 4B. The data isplotted as bound CPM on the Y-axis against the various potentialinhibitors and controls.

FIG. 24 shows the specificity of chicken-derived MMP-2 compositions foreither the integrin receptors α_(v)β₃ and α_(IIb)β₃ in the presence ofMMP-2 inhibitors as further described in Example 4B. The data ispresented as described in the legend in FIG. 23.

FIG. 25 show the effect of chicken MMP-2(410-637) GST fusion protein onbFGF-induced angiogenesis as described in Example 7A3). FIGS. 25A-B and25C-D respectively shown control (a non-MMP-2 fragment containing fusionprotein) and MMP-2 fragment GST fusion protein effects.

FIGS. 26 and 27 both illustrate in bar graph form the angiogenesis index(a measurement of branch points) of the effects of chickenMMP-2(410-637) GST fusion protein (labeled CTMMP-2) versus control(RAP-GST or GST-RAP) on bFGF-treated CAMs as described in Example 7A3).Angiogenic index is plotted on the Y-axis against the separatetreatments on the X-axis.

FIG. 28 shows the effects of peptides and organic compounds onbFGF-induced angiogenesis as measured by the effect on branch pointsplotted on the Y-axis against the various treatments on the X-axis,including bFGF alone, and bFGF-treated CAMs with peptides 69601 or 66203and organic compounds 96112, 96113 and 96229, as described in Examples7B and 14.

FIG. 29 graphically shows the dose response of peptide 85189 oninhibiting bFGF-induced angiogenesis as further described in Example7B2) where the number of branch points are plotted on the Y-axis againstthe amount of peptide administered to the embryo on the X-axis.

FIG. 30 shows the inhibitory activity of peptides 66203 (labeled 203)and 85189 (labeled 189) in bFGF-induced angiogenesis in the CAM assay asdescribed in Example 7B2). Controls included no peptide in bFGF-treatedCAMS and peptide 69601 (labeled 601). The data is plotted as describedin the legend for FIG. 27.

FIGS. 31A-L show the effect of various treatments against untreated CAMpreparations over a time course beginning at 24 hours and ending at 72hours as further described in Example 7B3). Photographs for thecategories labeled untreated, bFGF, bFGF+MAID (bFGF treated followedwith exposure to chicken MMP-2(2-4) GST fusion protein) and bFGF+control(bFGF treatment followed by chicken MMP-2(10-1) are respectively shownin FIGS. 31A-C, 31D-F, 31G-I, and 31J-L.

FIGS. 32, 33 and 34 respectively show the reduction in tumor weight forUCLAP-3, M21-L and FgM tumors following intravenous exposure to controlpeptide 69601 and antagonist 85189 as further described in Example 9A.The data is plotted with tumor weight on the Y-axis against the peptidetreatments on the X-axis.

FIG. 35 illustrates the effect of peptides and antibodies on melanomatumor growth in the chimeric mouse:human model as further described inExample 11B. The peptides assessed included control 69601 (labeled 601)and antagonist 85189 (labeled 189). The antibody tested was LM609. Tumorvolume in mm³ is plotted on the Y-axis against the various treatments onthe X-axis.

FIGS. 36A and B respectively show the effect of antagonist 85189(labeled 189) compared to control peptide 69601 (labeled 601) inreducing the volume and wet weight of M21L tumors over a dosage range of10, 50 and 250 ug/injection as further described in Example 11C.

FIGS. 37A and 37B show the effectiveness of antagonist peptide 85189(labeled 189 with a solid line and filled circles) against controlpeptide 69601 (labeled 601 on a dotted line and open squares) atinhibiting M21L tumor volume in the mouse:human model with two differenttreatment regimens as further described in Example 11C. Tumor volume inmm³ is plotted on the Y-axis against days on the X-axis.

FIGS. 38 through 42 schematically illustrate the various chemicalsyntheses of organic molecule α_(v)β₃ antagonists as further describedin Example 13.

FIGS. 43 and 44 show the effects of various organic molecules onbFGF-induced angiogenesis in a CAM assay as further described in Example14. Branch points are plotted on the Y-axis against the variouscompounds used at 250 ug/ml on the X-axis in FIG. 43 and 100 ug/ml inFIG. 44.

DETAILED DESCRIPTION OF THE INVENTION

A. Definitions

Amino Acid Residue: An amino acid formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the “L” isomeric form.However, residues in the “D” isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxy terminus of a polypeptide. In keeping withstandard polypeptide nomenclature (described in J. Biol. Chem.,243:3552-59 (1969) and adopted at 37 CFR §1.822 (b) (2)), abbreviationsfor amino acid residues are shown in the following Table ofCorrespondence:

TABLE OF CORRESPONDENCE SYMBOL 1-Letter 3-Letter AMINO ACID Y Tyrtyrosine G Gly glycine F Phe phenylalanine M Met methionine A Alaalanine S Ser serine I Ile isoleucine L Leu leucine T Thr threonine VVal valine P Pro proline K Lys lysine H His histidine Q Gln glutamine EGlu glutamic acid Z Glx Glu and/or Gln W Trp tryptophan R Arg arginine DAsp aspartic acid N Asn asparagine B Asx Asn and/or Asp C Cys cysteine XXaa unknown/other

In addition the following have the meanings below:

BOC tert-butyloxycarbonyl DCCI dicylcohexylcarbodiimide DMFdimethylformamide OMe methoxy HOBt 1-hydroxybezotriazole

It should be noted that all amino acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy-terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino acid residues.

Polypeptide: refers to a linear series of amino acid residues connectedto one another by peptide bonds between the alpha-amino group andcarboxy group of contiguous amino acid residues.

Peptide: as used herein refers to a linear series of no more than about50 amino acid residues connected one to the other as in a polypeptide.

Cyclic peptide: refers to a compound having a heteroatom ring structurethat includes several amide bonds as in a typical peptide. The cyclicpeptide can be a “head to tail” cyclized linear polypeptide in which alinear peptide's n-terminus has formed an amide bond with the terminalcarboxylate of the linear peptide, or it can contain a ring structure inwhich the polymer is homodetic or heterodetic and comprises amide bondsand/or other bonds to close the ring, such as disulfide bridges,thioesters, thioamides, guanidino, and the like linkages.

Protein: refers to a linear series of greater than 50 amino acidresidues connected one to the other as in a polypeptide.

Fusion protein: refers to a polypeptide containing at least twodifferent polypeptide domains operatively linked by a typical peptidebond (“fused”), where the two domains correspond to peptides no foundfused in nature.

Synthetic peptide: refers to a chemically produced chain of amino acidresidues linked together by peptide bonds that is free of naturallyoccurring proteins and fragments thereof.

B. General Considerations

The present invention relates generally to the discovery thatangiogenesis is mediated by the specific vitronectin receptor α_(v)β₃,and that inhibition of α_(v)β₃ function inhibits angiogenesis. Thisdiscovery is important because of the role that angiogenesis plays in avariety of disease processes. By inhibiting angiogenesis, one canintervene in the disease, ameliorate the symptoms, and in some casescure the disease.

Where the growth of new blood vessels is the cause of, or contributesto, the pathology associated with a disease, inhibition of angiogenesiswill reduce the deleterious effects of the disease. Examples includerheumatoid arthritis, diabetic retinopathy, inflammatory diseases,restenosis, and the like. Where the growth of new blood vessels isrequired to support growth of a deleterious tissue, inhibition ofangiogenesis will reduce the blood supply to the tissue and therebycontribute to reduction in tissue mass based on blood supplyrequirements. Examples include growth of tumors where neovascularizationis a continual requirement in order that the tumor grow beyond a fewmillimeters in thickness, and for the establishment of solid tumormetastases.

The methods of the present invention are effective in part because thetherapy is highly selective for angiogenesis and not other biologicalprocesses. As shown in the Examples, only new vessel growth containssubstantial α_(v)β₃, and therefore the therapeutic methods do notadversely effect mature vessels. Furthermore, α_(v)β₃ is not widelydistributed in normal tissues, but rather is found selectively on newvessels, thereby assuring that the therapy can be selectively targetedto new vessel growth.

The discovery that inhibition of α_(v)β₃ alone will effectively inhibitangiogenesis allows for the development of therapeutic compositions withpotentially high specificity, and therefore relatively low toxicity.Thus although the invention discloses the use of peptide-based reagentswhich have the ability to inhibit one or more integrins, one can designother reagents which more selectively inhibit α_(v)β₃, and therefore donot have the side effect of inhibiting other biological processes otherthat those mediated by α_(v)β₃.

For example, as shown by the present teachings, it is possible toprepare monoclonal antibodies highly selective for immunoreaction withα_(v)β₃ that are similarly selective for inhibition of α_(v)β₃ function.In addition, RGD-containing peptides can be designed to be selective forinhibition of α_(v)β₃, as described further herein.

Prior to the discoveries of the present invention, it was not known thatangiogenesis, and any of the processes dependent on angiogenesis, couldbe inhibited in vivo by the use of reagents that antagonize thebiological function of α_(v)β₃.

C. Methods for Inhibition of Angiogenesis

The invention provides for a method for the inhibition of angiogenesisin a tissue, and thereby inhibiting events in the tissue which dependupon angiogenesis. Generally, the method comprises administering to thetissue a composition comprising an angiogenesis-inhibiting amount of an60 _(v)β₃ antagonist.

As described earlier, angiogenesis includes a variety of processesinvolving neovascularization of a tissue including “sprouting”,vasculogenesis, or vessel enlargement, all of which angiogenesisprocesses are mediated by and dependent upon the expression of α_(v)β₃.With the exception of traumatic wound healing, corpus leuteum formationand embryogenesis, it is believed that the majority of angiogenesisprocesses are associated with disease processes and therefore the use ofthe present therapeutic methods are selective for the disease and do nothave deleterious side effects.

There are a variety of diseases in which angiogenesis is believed to beimportant, referred to as angiogenic diseases, including but not limitedto, inflammatory disorders such as immune and non-immune inflammation,chronic articular rheumatism and psoriasis, disorders associated withinappropriate or inopportune invasion of vessels such as diabeticretinopathy, neovascular glaucoma, restenosis, capillary proliferationin atherosclerotic plaques and osteoporosis, and cancer associateddisorders, such as solid tumors, solid tumor metastases, angiofibromas,retrolental fibroplasia, hemangiomas, Kaposi sarcoma and the likecancers which require neovascularization to support tumor growth.

Thus, methods which inhibit angiogenesis in a diseased tissueameliorates symptoms of the disease and, depending upon the disease, cancontribute to cure of the disease. In one embodiment, the inventioncontemplates inhibition of angiogenesis, per se, in a tissue. The extentof angiogenesis in a tissue, and therefore the extent of inhibitionachieved by the present methods, can be evaluated by a variety ofmethods, such as are described in the Examples for detectingα_(v)β₃-immunopositive immature and nascent vessel structures byimmunohistochemistry.

As described herein, any of a variety of tissues, or organs comprised oforganized tissues, can support angiogenesis in disease conditionsincluding skin, muscle, gut, connective tissue, joints, bones and thelike tissue in which blood vessels can invade upon angiogenic stimuli.

Thus, in one related embodiment, a tissue to be treated is an inflamedtissue and the angiogenesis to be inhibited is inflamed tissueangiogenesis where there is neovascularization of inflamed tissue. Inthis class the method contemplates inhibition of angiogenesis inarthritic tissues, such as in a patient with chronic articularrheumatism, in immune or non-immune inflamed tissues, in psoriatictissue and the like.

The patient treated in the present invention in its many embodiments isdesirably a human patient, although it is to be understood that theprinciples of the invention indicate that the invention is effectivewith respect to all mammals, which are intended to be included in theterm “patient”. In this context, a mammal is understood to include anymammalian species in which treatment of diseases associated withangiogenesis is desirable, particularly agricultural and domesticmammalian species.

In another related embodiment, a tissue to be treated is a retinaltissue of a patient with a retinal disease such as diabetic retinopathy,macular degeneration or neovascular glaucoma and the angiogenesis to beinhibited is retinal tissue angiogenesis where there isneovascularization of retinal tissue.

In an additional related embodiment, a tissue to be treated is a tumortissue of a patient with a solid tumor, a metastases, a skin cancer, abreast cancer, a hemangioma or angiofibroma and the like cancer, and theangiogenesis to be inhibited is tumor tissue angiogenesis where there isneovascularization of a tumor tissue. Typical solid tumor tissuestreatable by the present methods include lung, pancreas, breast, colon,laryngeal, ovarian, and the like tissues. Exemplary tumor tissueangiogenesis, and inhibition thereof, is described in the Examples.

Inhibition of tumor tissue angiogenesis is a particularly preferredembodiment because of the important role neovascularization plays intumor growth. In the absence of neovascularization of tumor tissue, thetumor tissue does not obtain the required nutrients, slows in growth,ceases additional growth, regresses and ultimately becomes necroticresulting in killing of the tumor.

Stated in other words, the present invention provides for a method ofinhibiting tumor neovascularization by inhibiting tumor angiogenesisaccording to the present methods. Similarly, the invention provides amethod of inhibiting tumor growth by practicing theangiogenesis-inhibiting methods.

The methods are also particularly effective against the formation ofmetastases because (1) their formation requires vascularization of aprimary tumor so that the metastatic cancer cells can exit the primarytumor and (2) their establishment in a secondary site requiresneovascularization to support growth of the metastases.

In a related embodiment, the invention contemplates the practice of themethod in conjunction with other therapies such as conventionalchemotherapy directed against solid tumors and for control ofestablishment of metastases. The administration of angiogenesisinhibitor is typically conducted during or after chemotherapy, althoughit is preferably to inhibit angiogenesis after a regimen of chemotherapyat times where the tumor tissue will be responding to the toxic assaultby inducing angiogenesis to recover by the provision of a blood supplyand nutrients to the tumor tissue. In addition, it is preferred toadminister the angiogenesis inhibition methods after surgery where solidtumors have been removed as a prophylaxis against metastases.

Insofar as the present methods apply to inhibition of tumorneovascularization, the methods can also apply to inhibition of tumortissue growth, to inhibition of tumor metastases formation, and toregression of established tumors. The Examples demonstrate regression ofan established tumor following a single intravenous administration of anα_(v)β₃ antagonist of this invention.

Restenosis is a process of smooth muscle cell (SMC) migration andproliferation at the site of percutaneous transluminal coronaryangioplasty which hampers the success of angioplasty. The migration andproliferation of SMC's during restenosis can be considered a process ofangiogenesis which is inhibited by the present methods. Therefore, theinvention also contemplates inhibition of restenosis by inhibitingangiogenesis according to the present methods in a patient followingangioplasty procedures. For inhibition of restenosis, the α_(v)β₃antagonist is typically administered after the angioplasty procedure forfrom about 2 to about 28 days, and more typically for about the first 14days following the procedure.

The present method for inhibiting angiogenesis in a tissue, andtherefore for also practicing the methods for treatment ofangiogenesis-related diseases, comprises contacting a tissue in whichangiogenesis is occurring, or is at risk for occurring, with acomposition comprising a therapeutically effective amount of an α_(v)β₃antagonist capable of inhibiting α_(v)β₃ binding to its natural ligand.Thus the method comprises administering to a patient a therapeuticallyeffective amount of a physiologically tolerable composition containingan α_(v)β₃ antagonist of the invention.

The dosage ranges for the administration of the α_(v)β₃ antagonistdepend upon the form of the antagonist, and its potency, as describedfurther herein, and are amounts large enough to produce the desiredeffect in which angiogenesis and the disease symptoms mediated byangiogenesis are ameliorated. The dosage should not be so large as tocause adverse side effects, such as hyperviscosity syndromes, pulmonaryedema, congestive heart failure, and the like. Generally, the dosagewill vary with the age, condition, sex and extent of the disease in thepatient and can be determined by one of skill in the art. The dosage canalso be adjusted by the individual physician in the event of anycomplication.

A therapeutically effective amount is an amount of α_(v)β₃ antagonistsufficient to produce a measurable inhibition of angiogenesis in thetissue being treated, ie., an angiogenesis-inhibiting amount. Inhibitionof angiogenesis can be measured in situ by immunohistochemistry, asdescribed herein, or by other methods known to one skilled in the art.

Insofar as an α_(v)β₃ antagonist can take the form of a α_(v)β₃ mimetic,an RGD-containing peptide, an anti-α_(v)β₃ monoclonal antibody, orfragment thereof, it is to be appreciated that the potency, andtherefore an expression of a “therapeutically effective” amount canvary. However, as shown by the present assay methods, one skilled in theart can readily assess the potency of a candidate α_(v)β₃ antagonist ofthis invention.

Potency of an α_(v)β₃ antagonist can be measured by a variety of meansincluding inhibition of angiogenesis in the CAM assay, in the in vivorabbit eye assay, in the in vivo chimeric mouse:human assay, and bymeasuring inhibition of binding of natural ligand to α_(v)β₃, all asdescribed herein, and the like assays.

A preferred α_(v)β₃ antagonist has the ability to substantially inhibitbinding of a natural ligand such as fibrinogen or vitronectin to α_(v)β₃in solution at antagonist concentrations of less than 0.5 micromolar(um), preferably less than 0.1 um, and more preferably less than 0.05um. By “substantially” is meant that at least a 50 percent reduction inbinding of fibrinogen is observed by inhibition in the presence of theα_(v)β₃ antagonist, and at 50% inhibition is referred to herein as anIC₅₀ value.

A more preferred α_(v)β₃ antagonist exhibits selectivity for α_(v)β₃over other integrins. Thus, a preferred α_(v)β₃ antagonist substantiallyinhibits fibrinogen binding to α_(v)β₃ but does not substantiallyinhibit binding of fibrinogen to another integrin, such as α_(v)β₁,α_(v)β₅ or α_(IIb)β₃. Particularly preferred is an α_(v)β₃ antagonistthat exhibits a 10-fold to 100-fold lower IC₅₀ activity at inhibitingfibrinogen binding to α_(v)β₃ compared to the IC₅₀ activity atinhibiting fibrinogen binding to another integrin. Exemplary assays formeasuring IC₅₀ activity at inhibiting fibrinogen binding to an integrinare described in the Examples.

A therapeutically effective amount of an α_(v)β₃ antagonist of thisinvention in the form of a monoclonal antibody is typically an amountsuch that when administered in a physiologically tolerable compositionis sufficient to achieve a plasma concentration of from about 0.01microgram (ug) per milliliter (ml) to about 100 ug/ml, preferably fromabout 1 ug/ml to about 5 ug/ml, and usually about 5 ug/ml. Stateddifferently, the dosage can vary from about 0.1 mg/kg to about 300mg/kg, preferably from about 0.2 mg/kg to about 200 mg/kg, mostpreferably from about 0.5 mg/kg to about 20 mg/kg, in one or more doseadministrations daily, for one or several days.

Where the antagonist is in the form of a fragment of a monoclonalantibody, the amount can readily be adjusted based on the mass of thefragment relative to the mass of the whole antibody. A preferred plasmaconcentration in molarity is from about 2 micromolar (uM) to about 5millimolar (mM) and preferably about 100 uM to 1 mM antibody antagonist.

A therapeutically effective amount of an α_(v)β₃ antagonist of thisinvention in the form of a polypeptide, or other similarly-sized smallmolecule α_(v)β₃ mimetic, is typically an amount of polypeptide suchthat when administered in a physiologically tolerable composition issufficient to achieve a plasma concentration of from about 0.1 microgram(ug) per milliliter (ml) to about 200 ug/ml, preferably from about 1ug/ml to about 150 ug/ml. Based on a polypeptide having a mass of about500 grams per mole, the preferred plasma concentration in molarity isfrom about 2 micromolar (uM) to about 5 millimolar (mM) and preferablyabout 100 uM to 1 mM polypeptide antagonist. Stated differently, thedosage per body weight can vary from about 0.1 mg/kg to about 300 mg/kg,and preferably from about 0.2 mg/kg to about 200 mg/kg, in one or moredose administrations daily, for one or several days.

The monoclonal antibodies or polypeptides of the invention can beadministered parenterally by injection or by gradual infusion over time.Although the tissue to be treated can typically be accessed in the bodyby systemic administration and therefore most often treated byintravenous administration of therapeutic compositions, other tissuesand delivery means are contemplated where there is a likelihood that thetissue targeted contains the target molecule. Thus, monoclonalantibodies or polypeptides of the invention can be administeredintravenously, intraperitoneally, intramuscularly, subcutaneously,intracavity, transdermally, and can be delivered by peristaltic means.

The therapeutic compositions containing a monoclonal antibody or apolypeptide of this invention are conventionally administeredintravenously, as by injection of a unit dose, for example. The term“unit dose” when used in reference to a therapeutic composition of thepresent invention refers to physically discrete units suitable asunitary dosage for the subject, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect in association with the required diluent; i.e.,carrier, or vehicle.

In one preferred embodiment as shown in the Examples, the α_(v)β₃antagonist is administered in a single dosage intravenously.

The compositions are administered in a manner compatible with the dosageformulation, and in a therapeutically effective amount. The quantity tobe administered and timing depends on the subject to be treated,capacity of the subject's system to utilize the active ingredient, anddegree of therapeutic effect desired. Precise amounts of activeingredient required to be administered depend on the judgement of thepractitioner and are peculiar to each individual. However, suitabledosage ranges for systemic application are disclosed herein and dependon the route of administration. Suitable regimes for administration arealso variable, but are typified by an initial administration followed byrepeated doses at one or more hour intervals by a subsequent injectionor other administration. Alternatively, continuous intravenous infusionsufficient to maintain concentrations in the blood in the rangesspecified for in viro therapies are contemplated.

As demonstrated by the present Examples, inhibition of angiogenesis andtumor regression occurs as early as 7 days after the initial contactingwith antagonist. Additional or prolonged exposure to antagonist ispreferable for 7 days to 6 weeks, preferably about 14 to 28 days.

In a related embodiment, the Examples demonstrate the relationshipbetween inhibition of α_(v)β₃ and induction of apoptosis in theneovasculature cells bearing α_(v)β₃. Thus, the invention alsocontemplates methods for inhibition of apoptosis in neovasculature of atissue. The method is practiced substantially as described herein forinhibition of angiogenesis in all tissues and conditions describedtherefor. The only noticeable difference is one of timing of effect,which is that apoptosis is manifest quickly, typically about 48 hoursafter contacting antagonist, whereas inhibition of angiogenesis andtumor regression is manifest more slowly, as described herein. Thisdifference affects the therapeutic regimen in terms of time ofadministration, and effect desired. Typically, administration forapoptosis of neovasculature can be for 24 hours to about 4 weeks,although 48 hours to 7 days is preferred.

D. Therapeutic Compositions

The present invention contemplates therapeutic compositions useful forpracticing the therapeutic methods described herein. Therapeuticcompositions of the present invention contain a physiologicallytolerable carrier together with an α_(v)β₃ antagonist as describedherein, dissolved or dispersed therein as an active ingredient. In apreferred embodiment, the therapeutic α_(v)β₃ antagonist composition isnot immunogenic when administered to a mammal or human patient fortherapeutic purposes.

As used herein, the terms “pharmaceutically acceptable”,“physiologically tolerable” and grammatical variations thereof, as theyrefer to compositions, carriers, diluents and reagents, are usedinterchangeably and represent that the materials are capable ofadministration to or upon a mammal without the production of undesirablephysiological effects such as nausea, dizziness, gastric upset and thelike.

The preparation of a pharmacological composition that contains activeingredients dissolved or dispersed therein is well understood in the artand need not be limited based on formulation. Typically suchcompositions are prepared as injectables either as liquid solutions orsuspensions, however, solid forms suitable for solution, or suspensions,in liquid prior to use can also be prepared. The preparation can also beemulsified.

The active ingredient can be mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredientand in amounts suitable for use in the therapeutic methods describedherein. Suitable excipients are, for example, water, saline, dextrose,glycerol, ethanol or the like and combinations thereof. In addition, ifdesired, the composition can contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agentsand the like which enhance the effectiveness of the active ingredient.

The therapeutic composition of the present invention can includepharmaceutically acceptable salts of the components therein.Pharmaceutically acceptable salts include the acid addition salts(formed with the free amino groups of the polypeptide) that are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, tartaric, mandelic and the like.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as, for example, sodium, potassium, ammonium,calcium or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like.

Particularly preferred are the salts of TFA and HCl, when used in thepreparation of cyclic polypeptide α_(v)β₃ antagonists. Representativesalts of peptides are described in the Examples.

Physiologically tolerable carriers are well known in the art. Exemplaryof liquid carriers are sterile aqueous solutions that contain nomaterials in addition to the active ingredients and water, or contain abuffer such as sodium phosphate at physiological pH value, physiologicalsaline or both, such as phosphate-buffered saline. Still further,aqueous carriers can contain more than one buffer salt, as well as saltssuch as sodium and potassium chlorides, dextrose, polyethylene glycoland other solutes.

Liquid compositions can also contain liquid phases in addition to and tothe exclusion of water. Exemplary of such additional liquid phases areglycerin, vegetable oils such as cottonseed oil, and water-oilemulsions.

A therapeutic composition contains an angiogenesis-inhibiting amount ofan α_(v)β₃ antagonist of the present invention, typically formulated tocontain an amount of at least 0.1 weight percent of antagonist perweight of total therapeutic composition. A weight percent is a ratio byweight of inhibitor to total composition. Thus, for example, 0.1 weightpercent is 0.1 grams of inhibitor per 100 grams of total composition.

E. Antagonists of Integrin α_(v)β₃

α_(v)β₃ antagonists are used in the present methods for inhibitingangiogenesis in tissues, and can take a variety of forms that includecompounds which interact with α_(v)β₃ in a manner such that functionalinteractions with natural α_(v)β₃ ligands are interfered. Exemplaryantagonists include analogs of α_(v)β₃ derived from the ligand bindingsite on α_(v)β₃, mimetics of either α_(v)β₃ or a natural ligand ofα_(v)β₃ that mimic the structural region involved in α_(v)β₃-ligandbinding interactions, polypeptides having a sequence corresponding to afunctional binding domain of the natural ligand specific for α_(v)β₃,particularly corresponding to the RGD-containing domain of a naturalligand of α_(v)β₃, and antibodies which immunoreact with either α_(v)β₃or the natural ligand, all of which exhibit antagonist activity asdefined herein.

1. Polypeptides

In one embodiment, the invention contemplates α_(v)β₃ antagonists in theform of polypeptides. A polypeptide (peptide) α_(v)β₃ antagonist canhave the sequence characteristics of either the natural ligand ofα_(v)β₃ or α_(v)β₃ itself at the region involved in α_(v)β₃-ligandinteraction and exhibits α_(v)β₃ antagonist activity as describedherein. A preferred α_(v)β₃ antagonist peptide contains the RGDtripeptide and corresponds in sequence to the natural ligand in theRGD-containing region.

Preferred RGD-containing polypeptides have a sequence corresponding tothe amino acid residue sequence of the RGD-containing region of anatural ligand of α_(v)β₃ such as fibrinogen, vitronectin, vonWillebrand factor, laminin, thrombospondin, and the like ligands. Thesequence of these α_(v)β₃ ligands are well known. Thus, an α_(v)β₃antagonist peptide can be derived from any of the natural ligands,although fibrinogen and vitronectin are preferred.

A particularly preferred α_(v)β₃ antagonist peptide preferentiallyinhibits α_(v)β₃ binding to its natural ligand(s) when compared to otherintegrins, as described earlier. These α_(v)β₃-specific peptides areparticularly preferred at least because the specificity for α_(v)β₃reduces the incidence of undesirable side effects such as inhibition ofother integrins. The identification of preferred α_(v)β₃ antagonistpeptides having selectivity for α_(v)β₃ can readily be identified in atypical inhibition of binding assay, such as the ELISA assay describedin the Examples.

A polypeptide of the present invention typically comprises no more thanabout 100 amino acid residues, preferably no more than about 60residues, more preferably no more than about 30 residues. Peptides canbe linear or cyclic, although particularly preferred peptides arecyclic.

Where the polypeptide is greater than about 100 residues, it istypically provided in the form of a fusion protein or protein fragment,as described herein.

Preferred cyclic and linear peptides and their designations are shown inTable 1 in the Examples.

It should be understood that a subject polypeptide need not be identicalto the amino acid residue sequence of a α_(v)β₃ natural ligand, so longas it includes the required sequence and is able to function as anα_(v)β₃ antagonist in an assay such as those described herein.

A subject polypeptide includes any analog, fragment or chemicalderivative of a polypeptide whose amino acid residue sequence is shownherein so long as the polypeptide is an α_(v)β₃ antagonist. Therefore, apresent polypeptide can be subject to various changes, substitutions,insertions, and deletions where such changes provide for certainadvantages in its use. In this regard, α_(v)β₃ antagonist polypeptide ofthis invention corresponds to, rather than is identical to, the sequenceof a recited peptide where one or more changes are made and it retainsthe ability to function as an α_(v)β₃ antagonist in one or more of theassays as defined herein.

Thus, a polypeptide can be in any of a variety of forms of peptidederivatives, that include amides, conjugates with proteins, cyclicpeptides, polymerized peptides, analogs, fragments, chemically modifiedpeptides, and the like derivatives.

The term “analog” includes any polypeptide having an amino acid residuesequence substantially identical to a sequence specifically shown hereinin which one or more residues have been conservatively substituted witha functionally similar residue and which displays the α_(v)β₃ antagonistactivity as described herein. Examples of conservative substitutionsinclude the substitution of one non-polar (hydrophobic) residue such asisoleucine, valine, leucine or methionine for another, the substitutionof one polar (hydrophilic) residue for another such as between arginineand lysine, between glutamine and asparagine, between glycine andserine, the substitution of one basic residue such as lysine, arginineor histidine for another, or the substitution of one acidic residue,such as aspartic acid or glutamic acid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residueprovided that such polypeptide displays the requisite inhibitionactivity.

A “chemical derivative” refers to a subject polypeptide having one ormore residues chemically derivatized by reaction of a functional sidegroup. In addition to side group derivitations, a chemical derivativecan have one or more backbone modifications including α-aminosubstitutions such as N-methyl, N-ethyl, N-propyl and the like, andα-carbonyl substitutions such as thioester, thioamide, guanidino and thelike. Such derivatized molecules include for example, those molecules inwhich free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those peptides which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For examples: 4-hydroxyproline may be substitutedfor praline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine.Polypeptides of the present invention also include any polypeptidehaving one or more additions and/or deletions or residues relative tothe sequence of a polypeptide whose sequence is shown herein, so long asthe requisite activity is maintained.

A particularly preferred derivative is a cyclic peptide according to theformula cyclo(Arg-Gly-Asp-D-Phe-NMeVal), abbreviated c(RGDf-NMeV), inwhich there is an N-methyl substituted α-amino group on the valineresidue of the peptide and cyclization has joined the primary amino andcarboxy termini of the peptide.

The term “fragment” refers to any subject polypeptide having an aminoacid residue sequence shorter than that of a polypeptide whose aminoacid residue sequence is shown herein.

When a polypeptide of the present invention has a sequence that is notidentical to the sequence of an α_(v)β₃ natural ligand, it is typicallybecause one or more conservative or non-conservative substitutions havebeen made, usually no more than about 30 number percent, and preferablyno more than 10 number percent of the amino acid residues aresubstituted. Additional residues may also be added at either terminus ofa polypeptide for the purpose of providing a “linker” by which thepolypeptides of this invention can be conveniently affixed to a label orsolid matrix, or carrier.

Labels, solid matrices and carriers that can be used with thepolypeptides of this invention are described hereinbelow.

Amino acid residue linkers are usually at least one residue and can be40 or more residues, more often 1 to 10 residues, but do not formα_(v)β₃ ligand epitopes. Typical amino acid residues used for linkingare tyrosine, cysteine, lysine, glutamic and aspartic acid, or the like.In addition, a subject polypeptide can differ, unless otherwisespecified, from the natural sequence of an α_(v)β₃ ligand by thesequence being modified by terminal-NH₂ acylation, e.g., acetylation, orthioglycolic acid amidation, by terminal-carboxylamidation, e.g., withammonia, methylamine, and the like terminal modifications. Terminalmodifications are useful, as is well known, to reduce susceptibility byproteinase digestion, and therefore serve to prolong half life of thepolypeptides in solutions, particularly biological fluids whereproteases may be present. In this regard, polypeptide cyclization isalso a useful terminal modification, and is particularly preferred alsobecause of the stable structures formed by cyclization and in view ofthe biological activities observed for such cyclic peptides as describedherein.

Any peptide of the present invention may be used in the form of apharmaceutically acceptable salt. Suitable acids which are capable offorming salts with the peptides of the present invention includeinorganic acids such as trifluoroacetic acid (TFA) hydrochloric acid(HCl), hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,sulfuric acid, methane sulfonic acid, acetic acid, phosphoric aceticacid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalicacid, malonic acid, succinic acid, maleic acid, fumaric acid,anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilicacid or the like. HCl and TFA salts are particularly preferred.

Suitable bases capable of forming salts with the peptides of the presentinvention include inorganic bases such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide and the like; and organic bases such asmono-, di- and tri-alkyl and aryl amines (e.g. triethylamine,diisopropyl amine, methyl amine, dimethyl amine and the like) andoptionally substituted ethanolamines (e.g. ethanolamine, diethanolamineand the like).

In addition, a peptide of this invention can be prepared as described inthe Examples without including a free ionic salt in which the chargedacid or base groups present in the amino acid residue side groups (e.g.,Arg, Asp, and the like) associate and neutralize each other to form an“inner salt” compound.

A peptide of the. present invention also referred to herein as a subjectpolypeptide, can be synthesized by any of the techniques that are knownto those skilled in the polypeptide art, including recombinant DNAtechniques. Synthetic chemistry techniques, such as a solid-phaseMerrifield-type synthesis, are preferred for reasons of purity,antigenic specificity, freedom from undesired side products, ease ofproduction and the like. An excellent summary of the many techniquesavailable can be found in Steward et al., “Solid Phase PeptideSynthesis”, W. H. Freeman Co., San Francisco, 1969; Bodanszky, et al.,“Peptide Synthesis”, John Wiley & Sons, Second Edition, 1976; J.Meienhofer, “Hormonal Proteins and Peptides”, Vol. 2, p. 46, AcademicPress (New York), 1983; Merrifield, Adv. Enzymol., 32:221-96, 1969;Fields et al., Int. J. Peptide Protein Res., 35:161-214, 1990; and U.S.Pat. No. 4,244,946 for solid phase peptide synthesis, and Schroder etal., “The Peptides”, Vol. 1, Academic Press (New York), 1965 forclassical solution synthesis, each of which is incorporated herein byreference. Appropriate protective groups usable in such synthesis aredescribed in the above texts and in J. F. W. McOmie, “Protective Groupsin Organic Chemistry”, Plenum Press, New York, 1973, which isincorporated herein by reference.

In general, the solid-phase synthesis methods contemplated comprise thesequential addition of one or more amino acid residues or suitablyprotected amino acid residues to a growing peptide chain. Normally,either the amino or carboxyl group of the first amino acid residue isprotected by a suitable, selectively removable protecting group. Adifferent, selectively removable protecting group is utilized for aminoacids containing a reactive side group such as lysine.

Using a solid phase synthesis as exemplary, the protected or derivatizedamino acid is attached to an inert solid support through its unprotectedcarboxyl or amino group. The protecting group of the amino or carboxylgroup is then selectively removed and the next amino acid in thesequence having the complimentary (amino or carboxyl) group suitablyprotected is admixed and reacted under conditions suitable for formingthe amide linkage with the residue already attached to the solidsupport. The protecting group of the amino or carboxyl group is thenremoved from this newly added amino acid residue, and the next aminoacid (suitably protected) is then added, and so forth. After all thedesired amino acids have been linked in the proper sequence, anyremaining terminal and side group protecting groups (and solid support)are removed sequentially or concurrently, to afford the final linearpolypeptide.

The resultant linear polypeptides prepared for example as describedabove may be reacted to form their corresponding cyclic peptides. Anexemplary method for preparing a cyclic peptide is described by Zimmeret al., Peptides 1992, pp. 393-394, ESCOM Science Publishers, B.V.,1993. Typically, tertbutoxycarbonyl protected peptide methyl ester isdissolved in methanol and sodium hydroxide solution are added and theadmixture is reacted at 20° C. (20 C.) to hydrolytically remove themethyl ester protecting group. After evaporating the solvent, thetertbutoxycarbonyl protected peptide is extracted with ethyl acetatefrom acidified aqueous solvent. The tertbutoxycarbonyl protecting groupis then removed under mildly acidic conditions in dioxane cosolvent. Theunprotected linear peptide with free amino and carboxy termini soobtained is converted to its corresponding cyclic peptide by reacting adilute solution of the linear peptide, in a mixture of dichloromethaneand dimethylformamide, with dicyclohexylcarbodiimide in the presence of1-hydroxybenzotriazole and N-methylmorpholine. The resultant cyclicpeptide is then purified by chromatography.

Alternative methods for cyclic peptide synthesis are described byGurrath et al., Eur. J. Biochem.,210:911-921 (1992), and described inthe Examples.

In addition, the α_(v)β₃ antagonist can be provided in the form of afusion protein. Fusion proteins are proteins produced by recombinant DNAmethods as described herein in which the subject polypeptide isexpressed as a fusion with a second carrier protein such as aglutathione sulfhydryl transferase (GST) or other well known carrier.Preferred fusion proteins comprise an MMP-2 polypeptide describedherein. The preparation of a MMP-2 fusion protein is described in theExamples.

Particularly preferred peptides and derivative peptides for use in thepresent methods are c-(GrGDFV) (SEQ ID NO 4), c-(RGDfV) (SEQ ID NO 5),c-(RADfV) (SEQ ID NO 6), c-(RGDFv) (SEQ ID NO 7), c-(RGDf-NMeV)(SEQ IDNO 15) and linear peptide YTAECKPQVTRGDVF (SEQ ID NO 8), where “c-”indicates a cyclic peptide, the upper case letters are single lettercode for an L-amino acid and the lower case letters are single lettercode for D-amino acid. The amino acid residues sequence of thesepeptides are also shown in SEQ ID NOs 4, 5, 6, 7, 15 and 8,respectively.

Also preferred are polypeptides derived from MMP-2 described herein,having sequences shown in SEQ ID Nos 17-28 and 45.

2. Monoclonal Antibodies

The present invention describes, in one embodiment, α_(v)β₃ antagonistsin the form of monoclonal antibodies which immunoreact with α_(v)β₃ andinhibit α_(v)β₃ binding to its natural ligand as described herein. Theinvention also describes cell lines which produce the antibodies,methods for producing the cell lines, and methods for producing themonoclonal antibodies.

A monoclonal antibody of this invention comprises antibody moleculesthat 1) immunoreact with isolated α_(v)β₃, and 2) inhibit fibrinogenbinding to α_(v)β₃ Preferred monoclonal antibodies which preferentiallybind to α_(v)β₃ include a monoclonal antibody having the immunoreactioncharacteristics of mAb LM609, secreted by hybridoma cell line ATCC HB9537. The hybridoma cell line ATCC HB 9537 was deposited pursuant toBudapest Treaty requirements with the American Type Culture Collection(ATCC), 1301 Parklawn Drive, Rockville, Md., USA, on Sep. 15, 1987.

The term “antibody or antibody molecule” in the various grammaticalforms is used herein as a collective noun that refers to a population ofimmunoglobulin molecules and/or immunologically active portions ofimmunoglobulin molecules, i.e., molecules that contain an antibodycombining site or paratope.

An “antibody combining site” is that structural portion of an antibodymolecule comprised of heavy and light chain variable and hypervariableregions that specifically binds antigen.

Exemplary antibodies for use in the present invention are intactimmunoglobulin molecules, substantially intact immunoglobulin moleculesand those portions of an immunoglobulin molecule that contain theparatope, including those portions known in the art as Fab, Fab′,F(ab′)₂ and F(v), and also referred to as antibody fragments.

In another preferred embodiment, the invention contemplates a truncatedimmunoglobulin molecule comprising a Fab fragment derived from amonoclonal antibody of this invention. The Fab fragment, lacking Fcreceptor, is soluble, and affords therapeutic advantages in serum halflife, and diagnostic advantages in modes of using the soluble Fabfragment. The preparation of a soluble Fab fragment is generally knownin the immunological arts and can be accomplished by a variety ofmethods.

For example, Fab and F(ab′)₂ portions (fragments) of antibodies areprepared by the proteolytic reaction of papain and pepsin, respectively,on substantially intact antibodies by methods that are well known. Seefor example, U.S. Pat. No. 4,342,566 to Theofilopolous and Dixon. Fab′antibody portions are also well known and are produced from F(ab′)₂portions followed by reduction of the disulfide bonds linking the twoheavy chain portions as with mercaptoethanol, and followed by alkylationof the resulting protein mercaptan with a reagent such as iodoacetamide.An antibody containing intact immunoglobulin molecules are preferred,and are utilized as illustrative herein.

The phrase “monoclonal antibody” in its various grammatical forms refersto a population of antibody molecules that contain only one species ofantibody combining site capable of immunoreacting with a particularepitope. A monoclonal antibody thus typically displays a single bindingaffinity for any epitope with which it immunoreacts. A monoclonalantibody may therefore contain an antibody molecule having a pluralityof antibody combining sites, each immunospecific for a differentepitope, e.g., a bispecific monoclonal antibody.

A monoclonal antibody is typically composed of antibodies produced byclones of a single cell called a hybridoma that secretes (produces) onlyone kind of antibody molecule. The hybridoma cell is formed by fusing anantibody-producing cell and a myeloma or other self-perpetuating cellline. The preparation of such antibodies was first described by Kohlerand Milstein, Nature 256:495-497 (1975), which description isincorporated by reference. Additional methods are described by Zola,Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987).The hybridoma supernates so prepared can be screened for the presence ofantibody molecules that immunoreact with α_(v)β₃ and for inhibition ofα_(v)β₃ binding to natural ligands.

Briefly, to form the hybridoma from which the monoclonal antibodycomposition is produced, a myeloma or other self-perpetuating cell lineis fused with lymphocytes obtained from the spleen of a mammalhyperimmunized with a source of α_(v)β₃, such as α_(v) ₃ isolated fromM21 human melanoma cells as described by Cheresh et al., J. Biol. Chem.,262:17703-17711 (1987).

It is preferred that the myeloma cell line used to prepare a hybridomabe from the same species as the lymphocytes. Typically, a mouse of thestrain 129 GlX⁺ is the preferred mammal. Suitable mouse myelomas for usein the present invention include thehypoxanthine-aminopterin-thymidine-sensitive (HAT) cell linesP3X63-Ag8.653, and Sp2/0-Agl4 that are available from the American TypeCulture Collection, Rockville, Md., under the designations CRL 1580 andCRL 1581, respectively.

Splenocytes are typically fused with myeloma cells using polyethyleneglycol (PEG) 1500. Fused hybrids are selected by their sensitivity toHAT. Hybridomas producing a monoclonal antibody of this invention areidentified using the enzyme linked immunosorbent assay (ELISA) describedin the Examples.

A monoclonal antibody of the present invention can also be produced byinitiating a monoclonal hybridoma culture comprising a nutrient mediumcontaining a hybridoma that secretes antibody molecules of theappropriate specificity. The culture is maintained under conditions andfor a time period sufficient for the hybridoma to secrete the antibodymolecules into the medium. The antibody-containing medium is thencollected. The antibody molecules can then be further isolated by wellknown techniques.

Media useful for the preparation of these compositions are both wellknown in the art and commercially available and include syntheticculture media, inbred mice and the like. An exemplary synthetic mediumis Dulbeccots minimal essential medium (DMEM; Dulbecco et al., Virol.8:396, 1959) supplemented with 4.5 gm/1 glucose, 20 mM glutamine, and20% fetal calf serum. An exemplary inbred mouse strain is the Balb/c.

Other methods of producing a monoclonal antibody, a hybridoma cell, or ahybridoma cell culture are also well known. See, for example, the methodof isolating monoclonal antibodies from an immunological repertoire asdescribed by Sastry, et al., Proc. Natl. Acad. Sci. USA, 86:5728-5732(1989); and Huse et al., Science, 246:1275-1281 (1989).

Also contemplated by this invention is the hybridoma cell, and culturescontaining a hybridoma cell that produce a monoclonal antibody of thisinvention. Particularly preferred is the hybridoma cell line thatsecretes monoclonal antibody mAb LM609 designated ATCC HB 9537. mAbLM609 was prepared as described by Cheresh et al., J. Biol. Chem.,262:17703-17711 (1987), and its preparation is also described in theExamples.

The invention contemplates, in one embodiment, a monoclonal antibodythat has the immunoreaction characteristics of mAb LM609.

It is also possible to determine, without undue experimentation, if amonoclonal antibody has the same (i.e., equivalent) specificity(immunoreaction characteristics) as a monoclonal antibody of thisinvention by ascertaining whether the former prevents the latter frombinding to a preselected target molecule. If the monoclonal antibodybeing tested competes with the monoclonal antibody of the invention, asshown by a decrease in binding by the monoclonal antibody of theinvention in standard competition assays for binding to the targetmolecule when present in the solid phase, then it is likely that the twomonoclonal antibodies bind to the same, or a closely related, epitope.

Still another way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to pre-incubatethe monoclonal antibody of the invention with the target molecule withwhich it is normally reactive, and then add the monoclonal antibodybeing tested to determine if the monoclonal antibody being tested isinhibited in its ability to bind the target molecule. If the monoclonalantibody being tested is inhibited then, in all likelihood, it has thesame, or functionally equivalent, epitopic specificity as the monoclonalantibody of the invention.

An additional way to determine whether a monoclonal antibody has thespecificity of a monoclonal antibody of the invention is to determinethe amino acid residue sequence of the CDR regions of the antibodies inquestion. Antibody molecules having identical, or functionallyequivalent, amino acid residue sequences in their CDR regions have thesame binding specificity. Methods for sequencing polypeptides is wellknown in the art.

The immunospecificity of an antibody, its target molecule bindingcapacity, and the attendant affinity the antibody exhibits for theepitope, are defined by the epitope with which the antibodyimmunoreacts. The epitope specificity is defined at least in part by theamino acid residue sequence of the variable region of the heavy chain ofthe immunoglobulin the antibody, and in part by the light chain variableregion amino acid residue sequence.

Use of the term “having the binding specificity of” indicates thatequivalent monoclonal antibodies exhibit the same or similarimmunoreaction (binding) characteristics and compete for binding to apreselected target molecule.

Humanized monoclonal antibodies offer particular advantages over murinemonoclonal antibodies, particularly insofar as they can be usedtherapeutically in humans. Specifically, human antibodies are notcleared from the circulation as rapidly as “foreign” antigens, and donot activate the immune system in the same manner as foreign antigensand foreign antibodies. Methods of preparing “humanized” antibodies aregenerally well known in the art, and can readily be applied to theantibodies of the present invention.

Thus, the invention contemplates, in one embodiment, a monoclonalantibody of this invention that is humanized by grafting to introducecomponents of the human immune system without substantially interferingwith the ability of the antibody to bind antigen.

3. α_(v)β₃-Specific Mimetics

The present invention demonstrates that α_(v)β₃ antagonists generallycan be used in the present invention, which antagonists can includepolypeptides, antibodies and other molecules, designated “mimetics”,which have the capacity to interefere with avD3 function. Particularlypreferred are antagonists which specifically interfere with α_(v)β₃function, and do not interfere with function of other integrins.

In this context it is appreciated that a variety of reagents may besuitable for use in the present methods, so long as these reagentsposses the requisite biological activity. These reagents are genericallyreferred to a mimetics because they possess the ability to “mimic” abinding domain on either α_(v)β₃ or the α_(v)β₃ ligand involved in thefunctional interaction of the receptor and ligand, and thereby interferewith (i.e., inhibit) normal function.

An α_(v)β₃ mimetic is any molecule, other than an antibody orligand-derived peptide, which exhibits the above-described properties.It can be a synthetic peptide, an analog or derivative of a peptide, acompound which is shaped like the binding pocket of the above-describedbinding domain such as an organic mimetic molecule, or other molecule.

A preferred mimetic of this invention is an organic-based molecule andthus is referred to as organic mimetic. Particularly preferred organicmimetic molecules that function as α_(v)β₃ antagonists by being amimetic to a ligand of α_(v)β₃ are Compounds 7, 9, 10, 12, 14, 15, 16,17 and 18 as described in Example 10.

The design of an α_(v)β₃ mimetic can be conducted by any of a variety ofstructural analysis methods for drug-design known in the art, includingmolecular modelling, two-dimensional nuclear magnetic resonance (2-DNMR) analysis, x-ray crystallography, random screening of peptide,peptide analog or other chemical polymer or compound libraries, and thelike drug design methodologies.

In view of the broad structural evidence presented in the presentspecification which shows that an α_(v)β₃ antagonist can be a fusionpolypeptide (e.g., an MMP-2 fusion protein), a small polypeptide, acyclic peptide, a derivative peptide, an organic mimetic molecule, or amonoclonal antibody, that are diversely different chemical structureswhich share the functional property of selective inhibition of α_(v)β₃,the structure of a subject α_(v)β₃ antagonist useful in the presentmethods need not be so limited, but includes any α_(v)β₃ mimetic, asdefined herein.

F. Methods for Identifying Antagonists of α_(v)β₃

The invention also described assay methods for identifying candidateα_(v)β₃ antagonists for use according to the present methods. In theseassay methods candidate molecules are evaluated for their potency ininhibiting α_(v)β₃ binding to natural ligands, and furthermore areevaluated for their potency in inhibiting angiogenesis in a tissue.

The first assay measures inhibition of direct binding of natural ligandto α_(v)β₃, and a preferred embodiment is described in detail in theExamples. The assay typically measures the degree of inhibition ofbinding of a natural ligand, such as fibrinogen, to isolated α_(v)β₃ inthe solid phase by ELISA.

The assay can also be used to identify compounds which exhibitspecificity for α_(v)β₃ and do not inhibit natural ligands from bindingother integrins. The specificity assay is conducted by running parallelELISA assays where both α_(v)β₃ and other integrins are screenedconcurrently in separate assay chambers for their respective abilitiesto bind a natural ligand and for the candidate compound to inhibit therespective abilities of the integrins to bind a preselected ligand.Preferred screening assay formats are described in the Examples.

The second assay measures angiogenesis in the chick chorioallantoicmembrane (CAM) and is referred to as the CAM assay. The CAM assay hasbeen described in detail by others, and further has been used to measureboth angiogenesis and neovascularization of tumor tissues. See Ausprunket al., Am. J. Pathol., 79:597-618 (1975) and Ossonski et al., CancerRes., 40:2300-2309 (1980).

The CAM assay is a well recognized assay model for in vivo angiogenesisbecause neovascularization of whole tissue is occurring, and actualchick embryo blood vessels are growing into the CAM or into the tissuegrown on the CAM.

As demonstrated herein, the CAM assay illustrates inhibition ofneovascularization based on both the amount and extent of new vesselgrowth. Furthermore, it is easy to monitor the growth of any tissuetransplanted upon the CAM, such as a tumor tissue. Finally, the assay isparticularly useful because there is an internal control for toxicity inthe assay system. The chick embryo is exposed to any test reagent, andtherefore the health of the embryo is an indication of toxicity.

The third assay that measures angiogenesis is the in vivo rabbit eyemodel and is referred to as the rabbit eye assay. The rabbit eye assayhas been described in detail by others, and further has been used tomeasure both angiogenesis and neovascularization in the presence ofangiogenic inhibitors such as thalidomide. See D'Amato, et al., Proc.Natl, Acad. Sci. USA, 91:4082-4085 (1994).

The rabbit eye assay is a well recognized assay model for in vivoangiogenesis because the neovascularization process, exemplified byrabbit blood vessels growing from the rim of the cornea into the cornea,is easily visualized through the naturally transparent cornea of theeye. Additionally, both the extent and the amount of stimulation orinhibition of neovascularization or regression of neovascularization caneasily be monitored over time.

Finally, the rabbit is exposed to any test reagent, and therefore thehealth of the rabbit is an indication of toxicity of the test reagent.

The fourth assay measures angiogenesis in the chimeric mouse:human mousemodel and is referred to as the chimeric mouse assay. The assay has beendescribed in detail by others, and further has been described herein tomeasure angiogenesis, neovascularization, and regression of tumortissues. See Yan, et al., J. Clin. Invest., 91:986-996 (1993). Thechimeric mouse assay is a useful assay model for in vivo angiogenesisbecause the transplanted skin grafts closely resemble normal human skinhistologically and neovascularization of whole tissue is occurringwherein actual human blood vessels are growing from the grafted humanskin into the human tumor tissue on the surface of the grafted humanskin. The origin of the neovascularization into the human graft can bedemonstrated by immunohistochemical staining of the neovasculature withhuman-specific endothelial cell markers.

As demonstrated herein, the chimeric mouse assay demonstrates regressionof neovascularization based on both the amount and extent of regressionof new vessel growth. Furthermore, it is easy to monitor effects on thegrowth of any tissue transplanted upon the grafted skin, such as a tumortissue. Finally, the assay is useful because there is an internalcontrol for toxicity in the assay system. The chimeric mouse is exposedto any test reagent, and therefore the health of the mouse is anindication of toxicity.

G. Article of Manufacture

The invention also contemplates an article of manufacture which is alabelled container for providing an α_(v)β₃ antagonist of the invention.An article of manufacture comprises packaging material and apharmaceutical agent contained within the packaging material.

The pharmaceutical agent in an article of manufacture is any of theα_(v)β₃ antagonists of the present invention, formulated into apharmaceutically acceptable form as described herein according the thedisclosed indications. The article of manufacture contains an amount ofpharmaceutical agent sufficient for use in treating a conditionindicated herein, either in unit or multiple dosages.

The packaging material comprises a label which indicates the use of thepharmaceutical agent contained therein, e.g., for treating conditionsassisted by the inhibition of angiogenesis, and the like conditionsdisclosed herein. The label can further include instructions for use andrelated information as may be required for marketing. The packagingmaterial can include container(s) for storage of the pharmaceuticalagent.

As used herein, the term packaging material refers to a material such asglass, plastic, paper, foil, and the like capable of holding withinfixed means a pharmaceutical agent. Thus, for example, the packagingmaterial can be plastic or glass vials, laminated envelopes and the likecontainers used to contain a pharamaceutical composition including thepharmaceutical agent.

In preferred embodiments, the packaging material includes a label thatis a tangible expression describing the contents of the article ofmanufacture and the use of the pharmaceutical agent contained therein.

EXAMPLES

The following examples relating to this invention are illustrative andshould not, of course, be construed as specifically limiting theinvention. Moreover, such variations of the invention, now known orlater developed, which would be within the purview of one skilled in theart are to be considered to fall within the scope of the presentinvention hereinafter claimed.

1. Preparation of Synthetic Peptides

a. Synthesis Procedure

The linear and cyclic polypeptides listed in Table 1 were synthesizedusing standard solid-phase synthesis techniques as, for example,described by Merrifield, Adv. Enzymol., 32:221-96, (1969), and Fields,G. B. and Noble, R. L., Int. J. Peptide Protein Res., 35:161-214,(1990).

Two grams (g) of BOC-Gly-D-Arg-Gly-Asp-Phe-Val-OMe (SEQ ID NO 1) werefirst dissolved in 60 milliliters (ml) of methanol to which was added1.5 ml of 2 N sodium hydroxide solution to form an admixture. Theadmixture was then stirred for 3 hours at 20 degrees C. (20 C.). Afterevaporation, the residue was taken up in water, acidified to pH 3 withdiluted HCl and extracted with ethyl acetate. The extract was dried overNa₂SO₄, evaporated again and the resultantBOC-Gly-D-Arg-Gly-Asp-Phe-Val-OH (SEQ ID NO 2) was stirred at 20 C. for2 hours with 20 ml of 2 N HCl in dioxane. The resultant admixture wasevaporated to obtain H-Gly-D-Arg-Gly-Asp-Phe-Val-OH (SEQ ID NO 3) thatwas subsequently dissolved in a mixture of 1800 ml of dichloromethaneand 200 ml of dimethylformamide (DMF) followed by cooling to 0 C.Thereafter, 0.5 g of dicyclohexylcarbodiimide (DCCI), 0.3 g of1-hydroxybenzotriazole (HOBt) and 0.23 ml of N-methylmorpholine wereadded successively with stirring.

The resultant admixture was stirred for a further 24 hours at 0 C. andthen at 20 C. for another 48 hours. The solution was concentrated andtreated with a mixed bed ion exchanger to free it from salts. After theresulting resin was removed by filtration, the clarified solution wasevaporated and the residue was purified by chromatography resulting inthe recovery of cyclo(-Gly-D-Arg-Gly-Asp-Phe-Val) (SEQ ID NO 4). Thefollowing peptides, listed in Table 1 using single letter code aminoacid residue abbreviations and identified by a peptide numberdesignation, were obtained analogously: cyclo(Arg-Gly-Asp-D-Phe-Val)(SEQ ID NO 5); cyclo(Arg-Ala-Asp-D-Phe-Val) (SEQ ID NO 6);cyclo(Arg-D-Ala-Asp-Phe-Val) (SEQ ID NO 9); cyclo(Arg-Gly-Asp-Phe-D-Val)(SEQ ID NO 7); and cyclo (Arg-Gly-Asp-D-Phe-NMeVal) (methylation is atthe alpha-amino nitrogen of the amide bond of the valine residue) (SEQID NO 15).

A peptide designated as 66203, having an identical sequence to that ofpeptide 62184, only differed from the latter by containing the salt HClrather than the TFA salt present in 62184. The same is true for thepeptides 69601 and 62185 and for 85189 and 121974.

b. Alternate Synthesis Procedure

i. Synthesis of cyclo-(Arg-Gly-Asp-DPhe-NmeVal). TFA salt

Fmoc-Arg(Mtr)-Gly-Asp(OBut)-DPhe-NMeVal-ONa is synthesized usingsolid-phase Merrifield-type procedures by sequentially adding NMeVal,DPhe, Asp(OBut), Gly and Fmoc-Arg(Mtr) in a step-wise manner to a4-hydroxymethyl-phenoxymethyl-polystyrene resin (Wang type resin)(customary Merrifield-type methods of peptide synthesis are applied asdescribed in Houben-Weyl, l.c., Volume 15/II, Pages 1 to 806 (1974). Thepolystyrene resin and amino acid residues precursors are commerciallyavailable from Aldrich, Sigma or Fluka chemical companies). Aftercompletion of sequential addition of the amino acid residues, the resinis then eliminated from the peptide chain using a 1:1 mixture ofTFA/dichloromethane which provides theFmoc-Arg(Mtr)-Gly-Asp(OBut)-DPhe-NMeVal-OH product. The Fmoc group isthen removed with a 1:1 mixture of piperidine/DMF which provides thecrude Arg(Mtr)-Gly-Asp(OBut)-DPhe-NMeVal-OH precursor which is thenpurified by HPLC in the customary manner.

For cyclization, a solution of 0.6 g ofArg(Mtr)-Gly-Asp(OBut)-DPhe-NMeVal-OH (synthesized above) in 15 ml ofDMF (dimethylformamide; Aldrich) is diluted with 85 ml ofdichloromethane (Aldrich), and 50 mg of NaHCO₃ are added. After coolingin a dry ice/acetone mixture, 40 μl of diphenylphosphoryl azide(Aldrich) are added. After standing at room temperature for 16 hours,the solution is concentrated. The concentrate is gel-filtered (SephadexG10 column in isopropanol/water 8:2) and then purified by HPLC in thecustomary manner. Treatment with TFA (trifluoroacetic acid)/H₂O (98:2)gives cyclo-(Arg-Gly-Asp-DPhe-NmeVal)×TFA which is then purified by HPLCin the customary manner; RT=19.5; FAB-MS (M+H): 589.

ii. Synthesis of “Inner Salt”

TFA salt is removed from the above-produced cyclic peptide by suspendingthe cyclo-(Arg-Gly-Asp-DPhe-NmeVal)×TFA in water followed by evaporationunder vacuum to remove the TFA. The cyclic peptideformed is referred toas an “inner salt” and is designated cyclo-(Arg-Gly-Asp-DPhe-NMeVal).The term “inner salt” is used because the cyclic peptide contains twooppositely charged residues which intra-electronically counterbalanceeach other to form an overall noncharged molecule. One of the chargedresidues contains an acid moiety and the other charged residue containsan amino moiety. When the acid moiety and the amino moiety are in closeproximity to one another, the acid moiety can be deprotonated by theamino moiety which forms a carboxylate/ammonium salt species with anoverall neutral charge.

iii. HCl treatment to give cyclo-(Arg-Gly-Asp-DPhe-NMeVal)×HCl

80 mg of cyclo-(Arg-Gly-Asp-DPhe-NMeVal) are dissolved in 0.01 M HClfive to six times and freeze dried after each dissolving operation.Subsequent purification by HPLC givecyclo-(Arg-Gly-Asp-DPhe-NMeVal)×HCl; FAB-MS (M+H): 589.

iv. Methane sulfonic acid treatment to givecyclo-(Arg-Gly-Asp-DPhe-NMeVal)×MeSO₃H

80 mg of cyclo-(Arg-Gly-Asp-DPhe-NMeVal) are dissolved in 0.01 M MeSO₃H(methane sulfonic acid) five to six times and freeze dried after eachdissolving operation. Subsequent purification by HPLC givecyclo-(Arg-Gly-Asp-DPhe-NMeVal)×MeSO₃H; RT=17.8 ; FAB-MS (M+H): 589.

Alternative methods of cyclization include derivatizing the side groupchains of an acyclic peptide precursor with sulfhydryl moieties, andwhen exposed to slightly higher than normal physiological pH conditions(pH 7.5), intramolecularly forms disulfide bonds with other sulfhydrylgroups present in the molecule to form a cyclic peptide. Additionally,the C-terminus carboxylate moiety of an acyclic peptide precurosor canbe reacted with a free sulfhydryl moiety present within the molecule forproducing thioester cyclized peptides.

In inhibition of angiogenesis assays as described in Example 7 where thesynthetic peptides were used, the 66203 peptide in HCl was slightly moreeffective in inhibiting angiogenesis than the identical peptide in TFA.

TABLE 1 Peptide Designation Amino Acid Sequence SEQ ID NO 62181cyclo(GrGDFV) 4 62184 (66203*) cyclo(RGDfV) 5 62185 (69601*)cyclo(RADfV) 6 62187 cyclo(RGDFv) 7 62880 YTAECKPQVTRGDVF 8 62186cyclo(RaDFV) 9 62175 cyclo(ARGDfL) 10 62179 cyclo(GRGDfL) 11 62411TRQVVCDLGNPM 12 62503 GVVRNNEALARLS 13 62502 TDVNGDGRHDL 14 121974(85189*) cyclo (RDGf-NH₂Me-V) 15 112784 cyclo (RGEf-NH₂Me-V) 16 huMMP-2(410-631)** 17 huMMP-2 (439-631)** 18 huMMP-2 (439-512)** 19 huMMP-2(439-546)** 20 huMMP-2 (510-631)** 21 huMMP-2 (543-631)** 22 chMMP-2(410-637)*** 23 chMMP-2 (445-637)*** 24 chMMP-2 (445-518)*** 25 chMMP-2(445-552)*** 26 chMMP-2 (516-637)*** 27 chMMP-2 (549-637)*** 28 *Thepeptides designated with an asterisk are prepared in HCl and areidentical in sequence to the peptide designated on the same line; thepeptides without an asterisk are prepared in TFA. Lower case lettersindicate a D-amino acid; capital letters indicate a L-amino acid. **Thehuman MMP-2 amino acid residue sequences for synthetic peptides areindicated by the corresponding residue positions shown in FIGS. 22A and22B. (MMP-2 refers to a member of the family of matrix metalloproteinaseenzymes). The human MMP-2 sequences are listed with the natural cysteineresidues but are not listed with engineered cysteine residues asdescribed for the fusion peptides. The non-natural cysteine residueswere substituted for the natural amino acid residue # at the indicatedresidue positions in order to facilitate solubility of the synthetic aswell as expressed fusion proteins and to ensure proper folding forpresentation of the binding site. ***The chicken MMP-2 amino acidresidue sequences for synthetic peptides are indicated by thecorresponding residue positions shown in FIGS. 22A and 22B. The chickenMMP-2 sequences are listed with the natural cysteine residues but notwith the engineered cysteine residues as described for the fusionpeptides as described above.

2. Monoclonal Antibodies

The monoclonal antibody LM609 secreted by hybridoma ATCC HB 9537 wasproduced using standard hybridoma methods by immunization with isolatedα_(v)β₃ adsorbed onto Sepharose-lentil lectin beads. The α_(v)β₃ hadbeen isolated from human melanoma cells designated M21, and antibody wasproduced as described by Cheresh et al., J. Biol. Chem., 262:17703-17711(1987). M21 cells were provided by Dr. D. L. Morton (University ofCalifornia at Los Angeles, Calif.) and grown in suspension cultures inRPMI 1640 culture medium containing 2 mM L-glutamine, 50 mg/mlgentamicin sulfate and 10% fetal calf serum.

Monoclonal antibody LM609 has been shown to specifically immunoreactwith α_(v)β₃ complex, and not immunoreact with α_(v) subunit, with β₃subunit, or with other integrins.

3. Characterization of the Tissue Distribution of α_(v)β₃ Expression

A. Immunofluorescence with Anti-Integrin Receptor Antibodies

During wound healing, the basement membranes of blood vessels expressseveral adhesive proteins, including von Willebrand factor, fibronectin,and fibrin. In addition, several members of the integrin family ofadhesion receptors are expressed on the surface of cultured smoothmuscle and endothelial cells. See, Cheresh, Proc. Natl. Acad. Sci., USA,84:6471 (1987); Janat et al., J. Cell Physiol., 151:588 (1992); andCheng et al., J. Cell Physiol., 139:275 (1989). Among these integrins isα_(v)β₃, the endothelial cell receptor for von Willebrand factor,fibrinogen (fibrin), and fibronectin as described by Cheresh, Proc.Natl. Acad. Sci., USA, 84:6471 (1987). This integrin initiates acalcium-dependent signaling pathway leading to endothelial cellmigration, and therefore appears to play a fundamental role in vascularcell biology as described by Leavelsey et al., J. Cell Biol., 121:163(1993).

To investigate the expression of α_(v)β₃ during angiogenesis, humanwound granulation tissue or adjacent normal skin was obtained fromconsenting patients, washed with 1 ml of phosphate buffered saline andembedded in O.T.C medium (Tissue Tek). The embedded tissues were snapfrozen in liquid nitrogen for approximately 30 to 45 seconds. Six micronthick sections were cut from the frozen blocks on a cryostat microtomefor subsequent immunoperoxidase staining with antibodies specific foreither β₃ integrins (α_(v)β₃ or α_(IIb)β₃) or the β₁ subfamily ofintegrins.

The results of the staining of normal human skin and wound granulationtissue are shown in FIGS. 1A-1D. Monoclonal antibodies AP3 and LM534,directed to β₃ and β₁ integrins, respectively, were used forimmunohistochemical analysis of frozen sections. Experiments with tissuefrom four different human donors yielded identical results. Thephotomicrographs are shown at magnification of 300×.

The α_(v)β₃ integrin was abundantly expressed on blood vessels ingranulation tissue (FIG. 1B) but was not detectable in the dermis andepithelium of normal skin from the same donor (FIG. 1A). In contrast, β₁integrins were abundantly expressed on blood vessels and stromal cellsin both normal skin (FIG. 1C) and granulation tissue (FIG. 1D) and, aspreviously shown as described by Adams et al., Cell, 63:425 (1991), onthe basal cells within the epithelium.

B. Immunofluorescence with Anti-Lipand Antibodies

Additional sections of the human normal skin and granulation tissuesprepared above were also examined for the presence of the ligands forthe β₃ and β₁ integrins, von Willebrand factor and laminin,respectively. Von Willebrand factor localized to the blood vessels innormal skin (FIG. 2A) and granulation tissue (FIG. 2B), whereas lamininlocalized to all blood vessels as well as the epithelial basementmembrane in both tissue preparations (FIGS. 2C and 2D).

C. Distribution of Anti-α_(v)β₃ Antibodies on Cancer Tissue

In addition to the above analyses, biopsies of cancer tissue from humanpatients were also examined for the presence and distribution ofα_(v)β₃. The tissues were prepared as described in Example 1A with theexception that they were stained with monoclonal antibody LM609 preparedin Example 2 that is specific for the integrin receptor complex,α_(v)β₃. In addition, tumors were also prepared for microscopichistological analysis by fixing representative examples of tumors inBulins Fixative for 8 hours and serial sections cut and H&E stained.

The results of immunoperoxidase staining of bladder, colon breast andlung cancer tissues are shown in FIGS. 3A-3D, respectively. α_(v)β₃ isabundantly expressed only on the blood vessels present in the fourcancer biopsies analyzed and not on any other cells present in thetissue.

The results described herein thus show that the α_(v)β₃ integrinreceptor is selectively expressed in specific tissue types, namelygranulated, metastatic tissues and other tissues in which angiogenesisis occurring and not normal tissue where the formation of new bloodvessels has stopped. These tissues therefore provide an ideal target fortherapeutic aspects of this invention.

4. Identification of α_(v)β₃-Specific Synthetic Peptides Detected byInhibition of Cell Attachment and by a Ligand-Receptor Binding Assay

A. Inhibition of Cell Attachment

As one means to determine integrin receptor specificity of theantagonists of this invention, inhibition of cell attachment assays wereperformed as described below.

Briefly, CS-1 hamster melanoma cells lacking expression of α_(v)β₃ andα_(v)β₃ were first transfected with an plasmid for expressing the β₃subunit as previously described by Filardo et al., J. Cell Biol.,130:441-450 (1995). Specificity of potential α_(v)β₃ antagonists wasdetermined by the ability to block the binding of α_(v)β₃-expressingCS-1 cells to VN or laminin coated plates. As an example of a typicalassay, the wells were first coated with 10 ug/ml substrate overnight.After rinsing and blocking with 1% heat-denatured BSA in PBS at roomtemperature for 30 minutes, peptide 85189 (SEQ ID NO 15) over aconcentration range of 0.0001 uM to 100 uM, was separately mixed withCS-1 cells for applying to wells with a cell number of 50,000cells/well. After a 10-15 minute incubation at 37 C., the solutioncontaining the cells and peptides was discarded. The number of attachedcells was then determined following staining with 1% crystal violet.Cell associated crystal violet was eluted by the addition of 100microliters (ul) of 10% acetic acid. Cell adhesion was quantified bymeasuring the optical density of the eluted crystal violet at a wavelength of 600 nm.

FIG. 21 shows the result of a typical assay with an α_(v)β₃ antagonist,here peptide 85189. No inhibition was detected with the peptide onlaminin-coated surfaces. In contrast, complete inhibition of binding wasobtained on VN-coated surfaces with a peptide concentration of 10 uM orgreater, as shown with the dose-response curve.

Similar assays were performed with fusion proteins containing variousregions of the MMP-2 protein. The MMP-2-derived polypeptides includeregions of the C-terminus of MMP-2 active in the binding interactionwith α_(v)β₃ and thereby capable of inhibiting MMP-2 activation andassociated activities. These polypeptides are prepared either assynthetic polypeptides having a sequence derived from the C-terminaldomain of MMP-2 as described in Example 1 or as fusion proteinsincluding all or a portion of the C-terminal domain of MMP-2, preparedas described below. MMP-2 C-terminal molecules are presented for bothchicken and human specific sequences.

The chicken-derived MMP-2 C-terminal domain, also referred to as thehemopexin domain immediately contiguous with the hinge region, comprisesthe amino acid residues 445-637 of MMP-2. The complete nucleotide andencoded amino acid sequence of chicken MMP-2 is described below. Thehuman MMP-2 nucleotide and encoded amino acid sequence is also describedbelow. The C-terminal domain in the human MMP-2 that corresponds to thechicken region of 445-637 begin at amino acid residue 439 and ends with631 due to six missing residues from the human sequence as shown inFIGS. 22A and 22B. Both human- and chicken-derived C-terminal MMP-2synthetic peptides for use in practicing the methods of this inventionare listed in Table 1. The amino acid residue sequences of the syntheticpeptides are the same as those generated by the recombinant fusionprotein counterparts but without the GST fusion component. TheC-terminal MMP-2 fusion proteins derived from both chicken and human areprepared as described below.

A MMP-2 fusion protein is a chimeric polypeptide having a sequence ofMMP-2 C-terminal domain or a portion thereof fused (operatively linkedby covalent peptide bond) to a carrier (fusion) protein, such asglutathione sulfhydryl transferase (GST).

To amplify various regions of chicken and human MMP-2, primer sequenceswere designed based on the known respective cDNA sequences of chickenand human MMP-2. The complete top strand of the cDNA nucleotide sequenceof unprocessed chicken MMP-2, also referred to as progelatinase, isshown in FIGS. 22A and 22B along with the deduced amino acid sequenceshown on the second line (Aimes et al., Biochem. J., 300:729-736, 1994).The third and fourth lines of the figure respectively show the deducedamino acid sequence of human (Collier et al., J. Biol. Chem.,263:6579-6587 (1988)) and mouse MMP-2 (Reponen et al., J. Biol. Chem.,267:7856-7862 (1992)). Identical residues are indicated by dots whilethe differing residues are given by their one letter IUPAC lettering.Missing residues are indicated by a dash. The numbering of the aminoacid residues starts from the first residue of the proenzyme, with theresidues of the signal peptide being given negative numbers. Thenucleotide sequence is numbered accordingly. The putative initation oftranslation (ATG) is marked with three forward arrowheads and thetranslation termination signal (TGA) is indicated by an asterisk. Theamino terminal sequences for the chicken proenzyme and active enzyme arecontained with diamonds and single arrowheads. The chicken progelatinasenucleotide and amino acid residue sequences are listed together as SEQID NO 29 while the encoded amino acid residue sequence is listedseparately as SEQ ID NO 30.

Templates for generating amplified regions of chicken MMP-2 were eithera cDNA encoding the full-length mature chicken MMP-2 polypeptideprovided by Dr. J. P. Quigley of the State University of New York atStoney Brook, N.Y. or a cDNA generated from a total cellular RNAtemplate derived by standard techniques from an excised sample ofchicken chorioallantoic membrane tissue. For the latter, the cDNA wasobtained with MuLV reverse transcriptase and a downstream primerspecific for the 3′-terminal nucleotides, 5′ATTGAATTCTTCTACAGTTCA3′ (SEQID NO 31), the 5′ and 3′ ends of which was respectively complementary tonucleotides 1932-1912 of the published chick MMP-2 sequence. Reversetranscriptase polymerase chain reaction (RT-PCR) was performed accordingto the specifications of the manufacturer for the GeneAmp RNA PCR Kit(Perkin Elmer). The primer was also engineered to contain an internalEcoRI restriction site.

From either of the above-described cDNA templates, a number ofC-terminal regions of chicken MMP-2, each having the natural cysteineresidue at position 637 at the carboxy terminus, were obtained by PCRwith the 3′ primer listed above (SEQ ID NO 31) paired with one of anumber of 5′ primers listed below. The amplified regions encoded thefollowing MMP-2 fusion proteins, having sequences corresponding to theamino acid residue positions as shown in FIGS. 22A and 22B and alsolisted in SEQ ID NO 30: 1) 203-637; 2) 274-637; 3) 292-637; 4) 410-637;5) 445-637. Upstream or 5′ primers for amplifying each of the nucleotideregions for encoding the above-listed MMP-2 fusion proteins weredesigned to encode the polypeptide start sites 3′ to an engineered,i.e., PCR-introduced, internal BamHI restriction site to allow fordirectional ligation into either pGEX-1λT or pGEX-3X expression vectors.The 5′ primers included the following sequences, the 5′ and 3′ ends ofwhich correspond to the indicated 5′ and 3′ nucleotide positions of thechicken MMP-2 sequence as shown in FIGS. 22A and 22B (the amino acidresidue position start sites are also indicated for each primer): 1)Nucleotides 599-619, encoding a 203 start site 5′ATGGGATCCACTGCAAATTTC3′(SEQ ID NO 32); 2) Nucleotides 809-830, encoding a 274 start site5′GCCGGATCCATGACCAGTGTA3′ (SEQ ID NO 33); 3) Nucleotides 863-883,encoding a 292 start site 5′GTGGGATCCCTGAAGACTATG3′ (SEQ ID NO 34); 4)Nucleotides 1217-1237, encoding a 410 start 5′AGGGGATCCTTAAGGGGATTC3′(SEQ ID NO 35); and 5) Nucleotides 1325-1345, encoding a 445 start site5′CTCGGATCCTCTGCAAGCACG3′ (SEQ ID NO 36).

The indicated nucleotide regions of the template cDNA were subsequentlyamplified for 35 cycles (annealing temperature 55 C.) according to themanufacturer's instructions for the Expand High Fidelity PCR System(Boehringer Mannheim). The resulting PCR products were gel-purified,digested with BamHI and EcoRI restriction enzymes, and repurified beforeligation into either pGEX-1λT or pGEX-3X vector (Pharmacia Biotech,Uppsala, Sweden) which had been similarly digested as well asdephosphorylated prior to the ligation reaction. The choice of plasmidwas based upon the required reading frame of the amplification product.Competent E. coli strain BSJ72 or BL21 cells were transformed with theseparate constructs by heat shock. The resulting colonies were screenedfor incorporation of the respective MMP-2 fusion protein-encodingplasmid by PCR prior to dideoxy sequencing of positive clones to verifythe integrity of the introduced coding sequence. In addition,verification of incorporation of plasmid was confirmed by expression ofthe appropriately-sized GST-MMP-2 fusion protein.

Purification of each of the recombinant GST-MMP-2 fusion proteins wasperformed using IPTG-induced log-phase cultures essentially as describedby the manufacturer for the GST Gene Fusion System (Pharmacia Biotech).Briefly, recovered bacteria were lysed by sonication and incubated withdetergent prior to clarification and immobilization of the recombinantprotein on sepharose 4B-coupled glutathione (Pharmacia Biotech). Afterextensive washing, the immobilized fusion proteins were separatelyeluted from the affinity matrix with 10 mM reduced glutathione in 50 mMTris-HCl, pH 8.0, and dialyzed extensively against PBS to removeresidual glutathione prior to use.

Prior attempts to produce fusion proteins between chicken MMP-2 residues445 and 637 that only had one encoded cysteine residue resulted ininsoluble products. Therefore, in order to generate additional solubleMMP-2 fusion proteins derived from the C-terminal region that did notinclude an endogenous terminal cysteine residue as present in thepreviously-described fusion protein, nucleotide sequences wereintroduced into amplified MMP-2 regions to encode a cysteine residue ifnecessary depending on the particular fusion protein. A cysteine residueis naturally present in the chicken MMP-2 sequence at position 446 andat position 637. In the human sequence, these positions correspondrespectively to 440 and 631. Therefore, fusion proteins were designed tocontain engineered terminal cysteine residues at the amino- orcarboxy-terminus of the chicken MMP-2 sequences of interest so as toprovide for disulfide-bonding with the naturally occurring cysteine atthe other terminus, as required by the construct.

Oligonucleotide primers were accordingly designed to allow foramplification of chicken MMP-2 C-terminal regions for expression ofsoluble MMP-2/GST fusion proteins. Amplified chicken MMP-2 C-terminalregions included those for encoding amino acid residue positions445-518, 445-552, 516-637 and 549-637. For fusion proteins containingresidue 517, the naturally encoded tyrosine residue was substituted fora cysteine to allow for disulfide bonding with either cysteine atresidue position 446 or 637. For fusion proteins containing residue 551,the naturally encoded tryptophan residue was substituted for a cysteineto allow for disulfide bonding with either naturally encoded cysteine atresidue position 446 or 637.

Briefly, the pGEX-3X plasmid construct encoding the recombinantGST/MMP-2(410-637) fusion protein prepared above was used as a templatefor amplification according to the manufacturer's protocol for theExpand High Fidelity PCR Kit (Boehringer Mannheim) utilizing a set ofoligonucleotide primers whose design was based on the published chickenMMP-2 sequence (also shown in FIGS. 22A and 22B. One upstream primer,designed to encode a chicken MMP-2 protein start site at position 445after an engineered internal BamHI endonuclease restriction site forinsertion into the pGEX-3X GST vector, had the nucleotide sequence(5′CTCGGATCCTCTGCAAGCACG3′ (SEQ ID NO 37)). The 5′ and 3′ ends of theprimer respectively corresponded to positions 1325-1345 of the chickenMMP-2 sequence in the figure. Another upstream primer, designed toencode a chicken MMP-2 protein start site at position 516 after anengineered internal BamHI restriction site for insertion into thepGEX-1λT GST vector and to encode a cysteine residue at position 517,had the nucleotide sequence (5′GCAGGATCCGAGTGCTGGGTTTATAC3′ (SEQ ID NO38)). The 5′ and 3′ ends of the primer respectively corresponded topositions 1537-1562 of the chicken MMP-2 sequence. A third upstreamprimer, designed to encode a chicken MMP-2 protein start site atposition 549 following an engineered internal EcoRI endonucleaserestriction site for insertion into the pGEX-1λT GST vector and toencode a cysteine residue at position 551, had the nucleotide sequence(5′GCAGAATTCAACTGTGGCAGAAACAAG3′ (SEQ ID NO 39)). The 5′ and 3′ ends ofthe primer respectively corresponded to positions 1639-1665 of thechicken MMP-2 sequence.

These upstream primers were separately used with one of the followingdownstream primers listed below to produce the above-described regionsfrom the C-terminal domain of chicken MMP-2. A first downstream primer(antisense), designed to encode a chicken MMP-2 protein termination siteat position 518, to encode a cysteine residue at position 517, and tocontain an internal EcoRI endonuclease restriction site for insertioninto a GST vector, had the nucleotide sequence(5′GTAGAATTCCAGCACTCATTTCCTGC3′ (SEQ ID NO 40)). The 5′ and 3′ ends ofthe primer, written in the 5′-3′ direction, were respectivelycomplementary in part to positions 1562-1537 of the chicken MMP-2sequence. A second downstream primer, designed to encode a chicken MMP-2protein termination site at position 552, to encode a cysteine residueat position 551, and to contain an internal EcoRI endonucleaserestriction site for insertion into a GST vector, had the nucleotidesequence (5′TCTGAATTCTGCCACAGTTGAAGG3′ (SEQ ID NO 41)). The 5′ and 3′ends of the primer, written in the 5′ -3′ direction, were respectivelycomplementary in part to positions 1666-1643 of the chicken MMP-2sequence. A third downstream primer, designed to encode a chicken MMP-2protein termination site at position 637 and to contain an internalEcoRI endonuclease restriction site for insertion into a GST vector, hadthe nucleotide sequence (5′ATTGAATTCTTCTACAGTTCA3′ (SEQ ID NO 42)). The5′ and 3′ ends of the primer, written in the 5′-3′ direction, wererespectively complementary in part to positions 1932-1912 of the chickenMMP-2 sequence.

The regions of the chicken MMP-2 carboxy terminus bounded by the aboveupstream and downstream primers, used in particular combinations toproduce the fusion proteins containing at least one engineered cysteineresidue as described above, were separately amplified for 30 cycles withan annealing temperature of 55 C. according to the manufacturer'sinstructions for the Expand High Fidelity PCR System (BoehringerMannheim). The resulting amplification products were separatelypurified, digested with BamHI and or EcoRI restriction enzymes asnecessary, and repurified before ligation into the appropriate GSTfusion protein vector, either pGEX-3X or pGEX-1λT, as indicated above bythe reading frame of the upstream oligonucleotide primer. For ligatingthe amplified MMP-2 products, the vectors were similarly digested aswell as dephosphorylated prior to the ligation reaction. Competent E.coli strain BL21 cells were then separately transformed with theresultant MMP-2-containing vector constructs by heat shock. Resultingcolonies were then screened for incorporation of the appropriate fusionprotein-encoding plasmid by PCR and production of the appropriate sizedGST-fusion protein prior to dideoxy sequencing of positive clones toverify the integrity of the introduced coding sequence. Purification ofrecombinant GST fusion proteins were then performed using IPTG-inducedlog-phase cultures essentially as described above for producing theother GST-MMP-2 fusion proteins.

The results of inhibition of cell attachment assays with various chickenMMP-2 proteins as well as with other peptides indicate that intactMMP-2, the fusion protein CTMMP-2(2-4) from residues 445-637 and peptide66203 (SEQ ID NO 5) but not MMP-2 (1-445) and control peptide 69601inhibited β₃-expressing CS-1 cell adhesion to vitronectin but notlaminin, and thereby inhibited vitronectin receptor α_(v)β₃ binding tovitronectin by interfering with normal α_(v)β₃ binding activity. Othertested CTMMP-2 fusion proteins 7-1 from residues 274-637, 10-1 fromresidues 292-637 and 4-3 from residues 274-400 had less affect on celladhesion compared to 2-4.

In addition to the chicken MMP-2 GST-fusion proteins described above,two human MMP-2 GST fusion proteins were produced for expressing aminoacid regions 203-631 and 439-631 of the mature human MMP-2 proenzymepolypeptide. The indicated regions correspond respectively to chickenMMP-2 regions 20637 and 445-637. Human MMP-2-GST fusion proteins wereproduced by PCR as described above for the chicken MMP-2-GST fusionproteins utilizing a cDNA template that encoded the entire human MMP-2open reading frame provided by Dr. W. G. Stetler-Stevenson at theNational Cancer Institute, Bethesda, Md. Upstream 5′ primer sequenceswere designed based upon the previously published sequence of humanMMP-2 (Collier et al., J. Biol. Chem., 263:6579-6587 (1988) and toencode an introduced internal EcoRI restriction site to allow forinsertion of the amplified products into the appropriate expressionvector.

One upstream primer, designed to encode a human MMP-2 protein start siteat position 203 after an engineered internal EcoRI endonucleaserestriction site for insertion into the pGEX-1λT GST vector, had thenucleotide sequence (5′GATGAATTCTACTGCAAGTT3′ (SEQ ID NO 43)). The 5′and 3′ ends of the primer respectively corresponded to positions 685-704of the human MMP-2 open reading frame sequence. Another upstream primer,designed to encode a human MMP-2 protein start site at position 439after an engineered internal EcoRI restriction site for insertion intothe pGEX-IXT GST vector, had the nucleotide sequence(5′CACTGAATTCATCTGCAAACA3′ (SEQ ID NO 44)). The 5′ and 3′ ends of theprimer respectively corresponded to positions 1392 and 1412 of the humanMMP-2 open reading frame sequence.

Each of the above primers were used separately with a downstream primer,having 5′ and 3′ ends respectively complementary to bases 1998 and 1978of the human MMP-2 sequence that ends distal to the MMP-2 open readingframe and directs protein termination after amino acid residue 631. Theamplified products produced expressed fusion proteins containing humanMMP-2 amino acid residues 203-631 (SEQ ID NO 45) and 439-631 (SEQ ID NO18).

The resulting PCR products were purified, digested with EcoRI andrepurified for ligation into a pGEX-1λT plasmid that was similarlydigested and dephosphorylated prior to the ligation reaction. Cells weretransformed as described above.

Other human MMP-2 fusion proteins containing amino acid residues 410-631(SEQ ID NO 17), 439-512 (SEQ ID NO 19), 439-546 (SEQ ID NO 20), 510-631(SEQ ID NO 21) and 543-631 (SEQ ID NO 22) are also prepared as describedabove for use in the methods of this invention.

B. Ligand-Receptor Binding Assay

The synthetic peptides prepared in Example 1 along with the MMP-2 fusionproteins described above were further screened by measuring theirability to antagonize α_(v)β₃ and α_(IIb)β₃ receptor binding activity inpurified ligand-receptor binding assays. The method for these bindingstudies has been described by Barbas et al., Proc. Natl. Acad. Sci. USA.90:10003-10007 (1993), Smith et al., J. Biol. Chem., 265:11008-11013(1990), and Pfaff et al., J. Biol. Chem., 269:20233-20238 (1994), thedisclosures of which are hereby incorporated by reference.

Herein described is a method of identifying antagonists in aligand-receptor binding assay in which the receptor is immobilized to asolid support and the ligand and antagonist are soluble. Also describedis a ligand-receptor binding assay in which the ligand is immobilized toa solid support and the receptor and antagonists are soluble.

Briefly, selected purified integrins were separately immobilized inTitertek microtiter wells at a coating concentration of 50 nanograms(ng) per well. The purification of the receptors used in theligand-receptor binding assays are well known in the art and are readilyobtainable with methods familiar to one of ordinary skill in the art.After incubation for 18 hours at 4 C., nonspecific binding sites on theplate were blocked with 10 milligrams/milliliter (mg/ml) of bovine serumalbumin (BSA) in Tris-buffered saline. For inhibition studies, variousconcentrations of selected peptides from Table 1 were tested for theability to block the binding of ¹²⁵I-vitronectin or ¹²⁵I-fibrinogen tothe integrin receptors, α_(v)β₃ and α_(IIb)β₃. Although these ligandsexhibit optimal binding for a particular integrin, vitronectin forα_(v)β₃ and fibrinogen for α_(IIb)β₃, inhibition of binding studiesusing peptides to block the binding of fibrinogen to either receptorallowed for the accurate determination of the amount in micromoles (uM)of peptide necessary to half-maximally inhibit the binding of receptorto ligand. Radiolabeled ligands were used at concentrations of 1 nM andbinding was challenged separately with unlabeled synthetic peptides.

Following a three hour incubation, free ligand was removed by washingand bound ligand was detected by gamma counting. The data from theassays where selected cyclic peptides listed in Table 1 were used toinhibit the binding of receptors and radiolabeled fibrinogen toseparately immobilized α_(v)β₃ and α_(IIb)β₃ receptors were highlyreproducible with the error between data points typically below 11%. TheIC₅₀ data in micromoles (IC₅₀ uM) are expressed as the average ofduplicate data points±the standard deviation as shown in Table 2.

TABLE 2 Peptide No. α_(V)β₃ (IC₅₀ uM) α_(IIb)β₃ (IC₅₀ uM) 62181 1.96 ±0.62 14.95 ± 7.84  62184  0.05 ± 0.001 0.525 ± 0.10  62185 0.885 ± 0.16   100 ± 0.001 62187  0.05 ± 0.001   0.26 ± 0.056 62186 57.45 ± 7.84   100 ± 0.001 62175 1.05 ± 0.07 0.63 ± 0.18 62179 0.395 ± .21  0.055 ±0.007

Thus, the RGD-containing or RGD-derivatized cyclic peptides 62181,62184, 62185 and 62187, each having one D-amino acid residue, exhibitedpreferential inhibition of fibrinogen binding to the α_(v)β₃ receptor asmeasured by the lower concentration of peptide required for half-maximalinhibition as compared to that for the α_(IIb)β₃ receptor. In contrast,the other RGD-containing or RGD-derivatized cyclic peptides, 62186,62175 and 62179, were either not as effective in blocking fibrinogenbinding to α_(v)β₃ or exhibited preferential inhibition of fibrinogenbinding to α_(IIb)β₃ as compared to α_(v)β₃. These results areconsistent with those recently published by Pfaff, et al., J. Biol.Chem., 269:20233-20238 (1994) in which the cyclic peptide RGDFV (whereinF indicates a D-amino acid residue) specifically inhibited binding offibrinogen to the α_(v)β₃ integrin and not to the α_(IIb)β₃ or α₅β₁integrins. Similar inhibition of binding assays were performed withlinearized peptides having or lacking an RGD motif, the sequences ofwhich were derived from the α_(v) receptor subunit, α_(IIb) receptorsubunit or vitronectin ligand amino acid residue sequences. Thesequences of the linear peptides, 62880 (VN-derived amino acid residues35-49), 62411 (α_(v)-derived amino acid residues 676-687); 62503(α_(v)-derived amino acid residues 655-667) and 62502 (α_(IIb)-derivedamino acid residues 296-306), are listed in Table 1. Each of thesepeptides were used in separate assays to inhibit the binding of eithervitronectin (VN) or fibrinogen (FG) to either α_(IIb)β₃ or α_(v)β₃. TheIC₅₀ data in micromoles (IC₅₀ uM) of an individual assay for eachexperiment is shown in Table 3.

TABLE 3 α_(IIb)β₃ IC₅₀ (uM) α_(V)β₃ IC₅₀ (uM) Peptide No. FG VN FG VN62880 4.2 0.98 <0.1 0.5 62411 >100 >100 >100 >10062503 >100 >100 >100 >100 62502 90 5 >100 >100

The results of inhibition of ligand binding assays to selected integrinreceptors with linearized peptides show that only peptide 62880 waseffective at inhibiting the half-maximal binding of either FG or VN toα_(v)β₃ as measured by the lower concentration of peptide required forhalf-maximal inhibition as compared to α_(IIb)β₃ receptor. None of theother linearized peptides were effective at blocking ligand binding toα_(v)β₃ although peptide 62502 was effective at blocking VN binding toα_(IIb)β₃.

In other ligand receptor binding assays performed as described abovewith the exception that detection of binding or inhibition thereof waswith ELISA and peroxidase-conjugated goat anti-rabbit IgG, the ligandsVN, MMP-2 and fibronectin at a range of 5-50 ng/well and listed in theorder of effectiveness were shown to bind to immobilized α_(v)β₃receptor while collagen did not. In addition, the ability of peptides toinhibit the binding of either MMP-2 or VN to immobilized α_(v)β₃ wasassessed with peptides 69601 (SEQ ID NO 6) and 66203 (SEQ ID NO 5). Onlypeptide 66203 was effective at inhibiting the binding of eithersubstrate to the α_(v)β₃ receptor while the control peptide 69601 failedto have an effect with either ligand.

Specificity of MMP-2 binding to integrin receptors was confirmed with asolid phase receptor binding assay in which iodinated MMP-2 was shown tobind to α_(v)β₃ and not to α_(IIb)β₃ that had been immobilized on asolid phase (300 bound cpm versus approximately 10 bound CPM). Theability of an MMP-2 derived peptide or fusion protein to inhibit thespecific binding of MMP-2 to α_(v)β₃ was demonstrated in a comparableassay, the results of which are shown in FIG. 23. TheGST-CTMMP-2(445-637) (also referred to as CTMMP-2(2-4)) fusion proteinprepared as described above, labeled GST-MAID, inhibited the binding ofiodinated MMP-2 to α_(v)β₃ while GST alone had no effect with levels ofbound CPM comparable to wells receiving no inhibitor at all (labeledNT). The MMP-2 fusion protein referred to as CTMMP-2(274-637), alsoreferred to as CTMMP-2(10-1), failed to inhibit the binding of labeledMMP-2 to α_(v)β₃.

Specificity of receptor interaction with MMP-2-derived antagonists wasconfirmed with binding and inhibition of binding solid phase assays.CTMMP-2(2-4), labeled in FIG. 24 as [125I]GST2-4, bound to α_(v)β₃ andnot to α_(IIb)β₃ while CTMMP-2(10-1), labeled in FIG. 24 as[125I]GST10-1, did not bind to either receptor in the in vitro solidphase assay. In addition, the binding of labeled GST2-4 was competed byunlabeled GST2-4.

Thus, the ligand-receptor assay described herein can be used to screenfor both circular or linearized synthetic peptides that exhibitselective specificity for a particular integrin receptor, specificallyα_(v)β₃, as used as vitronectin receptor (α_(v)β₃) antagonists inpracticing this invention.

5. Characterization of the Untreated Chick Chorioallantoic Membrane(CAM)

A. Preparation of the CAM

Angiogenesis can be induced on the chick chorioallantoic membrane (CAM)after normal embryonic angiogenesis has resulted in the formation ofmature blood vessels. Angiogenesis has been shown to be induced inresponse to specific cytokines or tumor fragments as described byLeibovich et al., Nature, 329:630 (1987) and Ausprunk et al., Am. J.Pathol., 79:597 (1975). CAMs were prepared from chick embryos forsubsequent induction of angiogenesis and inhibition thereof as describedin Examples 6 and 7, respectively. Ten day old chick embryos wereobtained from McIntyre Poultry (Lakeside, Calif.) and incubated at 37 C.with 60% humidity. A small hole was made through the shell at the end ofthe egg directly over the air sac with the use of a small crafts drill(Dremel, Division of Emerson Electric Co. Racine Wis.). A second holewas drilled on the broad side of the egg in a region devoid of embryonicblood vessels determined previously by candling the egg. Negativepressure was applied to the original hole, which resulted in the CAM(chorioallantoic membrane) pulling away from the shell membrane andcreating a false air sac over the CAM. A 1.0 centimeter (cm)×1.0 cmsquare window was cut through the shell over the dropped CAM with theuse of a small model grinding wheel (Dremel). The small window alloweddirect access to the underlying CAM.

The resultant CAM preparation was then either used at 6 days ofembryogenesis, a stage marked by active neovascularization, withoutadditional treatment to the CAM reflecting the model used for evaluatingeffects on embryonic neovascularization or used at 10 days ofembryogenesis where angiogenesis has subsided. The latter preparationwas thus used in this invention for inducing renewed angiogenesis inresponse to cytokine treatment or tumor contact as described in Example6.

B. Histology of the CAM

To analyze the microscopic structure of the chick embryo CAMs and/orhuman tumors that were resected from the chick embryos as described inExample 8, the CAMs and tumors were prepared for frozen sectioning asdescribed in Example 3A. Six micron (um) thick sections were cut fromthe frozen blocks on a cryostat microtome for immunofluorescenceanalysis.

FIG. 4 shows a typical photomicrograph of an area devoid of bloodvessels in an untreated 10 day old CAM. As angiogenesis in the CAMsystem is subsiding by this stage of embryogenesis, the system is usefulin this invention for stimulating the production of new vasculature fromexisting vessels from adjacent areas into areas of the CAM currentlylacking any vessels.

C. Integrin Profiles in the CAM Detected by Immunofluorescence

To view the tissue distribution of integrin receptors present in CAMtissues, 6 um frozen sections of both tumor tissue and chick embryo CAMtissues were fixed in acetone for 30 seconds and stained byimmunofluorescence with 10 micrograms/milliliter (ug/ml) mAb CSAT, amonoclonal antibody specific for the , integrin subunit as described byBuck et al., J. Cell Biol., 107:2351 (1988) and thus used for controls,or LM609 as prepared in Example 2. Primary staining was followed bystaining with a 1:250 dilution of goat anti-mouse rhodamine labeledsecondary antibody (Tago) to allow for the detection of the primaryimmunoreaction product. The sections were then analyzed with a Zeissimmunofluorescence compound microscope.

The results of the immunofluorescence analysis show that the matureblood vessels present in an untreated 10 day chick embryo expressed theintegrin β₁ subunit (FIG. 5A). In contrast, in a serial section of thetissue shown in FIG. 5A, no immunoreactivity with LM609 was revealed(FIG. 5B). Thus, the integrin α_(v)β₃ detected by the LM609 antibody wasnot actively being expressed by the mature blood vessels present in a 10day old untreated chick embryo. As shown in the CAM model and in thefollowing Examples, while the blood vessels are undergoing new growth innormal embryogenesis or induced by either cytokines or tumors, the bloodvessels are expressing α_(v)β₃. However, following activeneovascularization, once the vessels have stopped developing, theexpression of α_(v)β₃ diminishes to levels not detectable byimmunofluorescence analysis. This regulation of α_(v)β₃ expression inblood vessels undergoing angiogenesis as contrasted to the lack ofexpression in mature vessels provides for the unique ability of thisinvention to control and inhibit angiogenesis as shown in the followingExamples using the CAM angiogenesis assay system.

In other profiles, the metalloproteinase MMP-2 and α_(v)β₃ colocalizedon endothelial cells undergoing angiogenesis three days following bFGFinduction in the 10 day old CAM model. MMP-2 was only minimallyexpressed on vessels that lacked the α_(v)β₃ receptor. In addition,MMP-2 colocalized with α_(v)β₃ on angiogenic M21-L tumor-associatedblood vessels in vivo (tumors resulting from injection of M21-L humanmelanoma cells into the dermis of human skin grafts grown on SCID miceas described in Example 11) but not with preexisting non-tumorassociated blood vessels. Similar results of the selective associationof MMP-2 and α_(v)β₃ were also obtained with α_(v)β₃ bearing CS-1melanoma tumors in the CAM model but not with CS-1 cells lackingα_(v)β₃.

6. CAM Anciogenesis Assay

A. Angiogenesis Induced by Growth Factors

Angiogenesis has been shown to be induced by cytokines or growth factorsas referenced in Example 5A. In the experiments described herein,angiogenesis in the CAM preparation described in Example 5 was inducedby growth factors that were topically applied onto the CAM blood vesselsas described herein.

Angiogenesis was induced by placing a 5 millimeter (mm)×5 mm Whatmanfilter disk (Whatman Filter paper No.1) saturated with Hanks BalancedSalt Solution (HBSS, GIBCO, Grand Island, N.Y.) or HBSS containing 150nanograms/milliliter (ng/ml) recombinant basic fibroblast growth factor(bFGF) (Genzyme, Cambridge, Mass.) on the CAM of a 10-day chick embryoin a region devoid of blood vessels and the windows were latter sealedwith tape. In other assays, 125 ng/ml bFGF was also effective atinducing blood vessel growth. For assays where inhibition ofangiogenesis ws evaluated with intravenous injections of antagonists,angiogenesis was first induced with 1-2 ug/ml bFGF in fibroblast growthmedium. Angiogenesis was monitored by photomicroscopy after 72 hours.CAMs were snap frozen, and 6 um cryostat sections were fixed withacetone and stained by immunofluorescence as described in Example 5Cwith 10 ug/ml of either anti-D₁ monoclonal antibody CSAT or LM609.

The immunofluorescence photomicrograph in FIG. 5C shows enhancedexpression of α_(v)β₃ during bFGF-induced angiogenesis on the chick CAMin contrast with the absence of α_(v)β₃ expression in an untreated chickCAM as shown in FIG. 5B. α_(v)β₃ was readily detectable on many (75% to80%) of the vessels on the bFGF-treated CAMs. In addition, theexpression of integrin β₁ did not change from that seen in an untreatedCAM as β₁ was also readily detectable on stimulated blood vessels.

The relative expression of α_(v)β₃ and β₁ integrins was then quantifiedduring bFGF-induced angiogenesis by laser confocal image analysis of theCAM cryostat sections. The stained sections were then analyzed with aZeiss laser confocal microscope. Twenty-five vessels stained with LM609and 15 stained with CSAT (average size ˜1200 sq mm², range 350 to 3,500mm²) were selected from random fields and the average rhodaminefluorescence for each vessel per unit area was measured in arbitraryunits by laser confocal image analysis. Data are expressed as the meanfluorescence intensity in arbitrary units of vessels±standard error(SE).

The results plotted in FIG. 6 show that staining of α_(v)β₃ wassignificantly enhanced (four times higher) on CAMs treated with bFGF asdetermined by the Wilcoxon Rank Sum Test (P<0.000l) whereas β₁ stainingwas not significantly different with bFGF treatment.

The CAM assay was further used to examine the effect of another potentangiogenesis inducer, tumor necrosis factor-alpha (TNFA), on theexpression of β₁ and β₃ integrins. Filter disks impregnated with eitherbFGF or TNFα and placed on CAMs from 10 day embryos were found topromote local angiogenesis after 72 hours.

The results are shown in the photomicrographs of CAMs either untreated(FIG. 7A), treated with bFGF (FIG. 7B) or treated with TNFα (FIG. 7C).Blood vessels are readily apparent in both the bFGF and TNFα treatedpreparations but are not present in the untreated CAM. Thus, the topicalapplication of a growth factor/cytokine resulted in the induction ofangiogenesis from mature vessels in an adjacent area into the areaoriginally devoid of blood vessels. In view of the bFGF-induced bloodvessels and concomitant expression of α_(v)β₃ as shown in FIG. 5C,treatment of TNFα results in comparable activities.

These findings indicate that in both human and chick, blood vesselsinvolved in angiogenesis show enhanced expression of α_(v)β₃. Consistentwith this, expression of α_(v)β₃ on cultured endothelial cells can beinduced by various cytokines in vitro as described by Janat et al., J.Cell Physiol., 151:588 (1992); Enenstein et al., Exp. Cell Res., 203:499(1992) and Swerlick et al., J. Invest. Derm., 99:715 (1993).

The effect on growth-factor induced angiogenesis by antibody and peptideinhibitors is presented in Examples 7A and 7B.

B. Embryonic Angioqenesis

The CAM preparation for evaluating the effect of angiogenesis inhibitorson the natural formation of embryonic neovasculature was the 6 dayembryonic chick embryo as previously described. At this stage indevelopment, the blood vessels are undergoing de novo growth and thusprovides a useful system for determining if α_(v)β₃ participates inembryonic angiogenesis. The CAM system was prepared as described abovewith the exception that the assay was performed at embryonic day 6rather than at day 10. The effect on embryonic angiogenesis by treatmentwith antibodies and peptides of this invention are presented in Example7C.

C. Angiogenesis Induced by Tumors

To investigate the role of α_(v)β₃ in tumor-induced angiogenesis,various α_(v)β₃-negative human melanoma and carcinoma fragments wereused in the CAM assay that were previously grown and isolated from theCAM of 17-day chick embryo as described by Brooks et al., J. Cell Biol.,122:1351 (1993) and as described herein. The fragments induced extensiveneovascularization in the presence of buffer alone.

Angiogenesis was induced in the CAM assay system by direct apposition ofa tumor fragment on the CAM. Preparation of the chick embryo CAM wasidentical to the procedure described above. Instead of a filter paperdisk, a 50 milligram (mg) to 55 mg in weight fragment of one of humanmelanoma tumor M21-L, human lung carcinoma tumor UCLAP-3, humanpancreatic carcinoma cell line FG (Cheresh et al., I Cell 58:945-953,1989), or human laryngeal carcinoma cell line HEp3, all of which areα_(v)β₃ negative tumors, was placed on the CAM in an area originallydevoid of blood vessels.

The M21-L human melanoma cell line, UCLAP-3 human lung carcinoma cellline, FG pancreatic carcinoma cell line, or HEp3 human laryngealcarcinoma cell line, all α_(v)β₃ negative, were used to grow the solidhuman tumors on the CAMs of chick embryos. A single cell suspension of8×10⁶ M21-L, UCLAP-3, and FB or 5×10⁵ HEp3 cells was first applied tothe CAMs in a total volume of 30 ul of sterile HBSS. The windows weresealed with tape and the embryos were incubated for 7 days to allowgrowth of human tumor lesions. At the end of 7 days, now a 17-dayembryo, the tumors were resected from the CAMs and trimmed free ofsurrounding CAM tissue. The tumors were sliced into 50 mg to 55 mg tumorfragments for use in either angiogenesis or tumor growth assays. Thetumor fragments were placed on a new set of 10 day chick embryo CAMs asdescribed in Example 6A in an area devoid of blood vessels.

Tumors grown in vivo on the chick embryo CAMs were stained for α_(v)β₃expression with mAb LM609 as described in Example 3A. No specificstaining of tumor cells was observed indicating a lack of α_(v)β₃expression.

These CAM tumor preparations were then subsequently treated as describedin Examples 7D and 7E for measuring the effects of antibodies andpeptides on tumor-induced angiogenesis. The CAM tumor preparations werealso treated as described in Examples 8, 9, and 12 for measuring theeffects of antibodies and peptides on regression of tumors and apoptosisof angiogenic blood vessels and vascular cells.

7. Inhibition of Angiogenesis as Measured in the CAM Assay

A. Inhibition of Growth Factor-Induced Angiogenesis by TopicalApplication of Inhibitors

1) Treatment with Monoclonal Antibodies

To determine whether α_(v)β₃ plays an active role in angiogenesis,filter disks saturated with bFGF or TNFα were placed on CAMs then themonoclonal antibodies (also referred to as mAb) , LM609 (specific forα_(v)β₃) CSAT (specific for β₁), or P3G2 or also P1F6 (both specific forα_(v)β₅) were added to the preparation.

Angiogenesis was induced on CAMs from 10 day chick embryos by filterdisks saturated with bFGF. Disks were then treated with 50 ml HBSScontaining 25 mg of mAb in a total volume of 25 ul of sterile HBSS at 0,24, and 48 hours. At 72 hours, CAMs were harvested and placed in a 35 mmpetri dish and washed once with 1 ml of phosphate buffered saline. Thebottom side of the filter paper and CAM tissue was then analyzed underan Olympus stereo microscope, with two observers in a double-blindfashion. Angiogenesis inhibition was considered significant when CAMsexhibited >50% reduction in blood vessel infiltration of the CAMdirectly under the disk. Experiments were repeated four times perantibody, with 6 to 7 embryos per condition.

The results of the effects of mAb treatment on bFGF-induced angiogenesisis shown in FIGS. 8A-8B. An untreated CAM preparation devoid of bloodvessels is shown in FIG. 8A to provide a comparison with the bFGF-bloodvessel induction shown in FIG. 8B and effects thereon by the mAbs inFIGS. 8C-8E. About 75% of these CAMs treated with mAb LM609exhibited >50% inhibition of angiogenesis as shown in FIG. 8E, and manyof these appeared devoid of vessel infiltration. In contrast, the buffercontrol (FIG. 8A) and disks treated with mAbs CSAT (FIG. 8C) and P3G2(FIG. 8D) consistently showed extensive vascularization.

Identical results were obtained when angiogenesis was induced with TNFα.To examine the effects of these same antibodies on preexisting matureblood vessels present from normal vessel development adjacent to theareas devoid of vessels, filter disks saturated with mAbs were placed onvascularized regions of CAMs from 10 day embryos that did not receivetopical application of cytokine. None of the three mAbs affectedpreexisting vessels, as assessed by visualization under a stereomicroscope. Thus, mAb LM609 selectively inhibited only new blood vesselgrowth and did not effect mature blood vessels present in adjacentareas. This same effect was seen with the application of syntheticpeptides either applied topically or intravenously as described inExamples 7A2) and 7E2), respectively.

2) Treatment with Synthetic Peptides

CAM assays were also performed with the synthetic peptides of thisinvention to determine the effect of cyclic and linearized peptides ongrowth factor induced angiogenesis. The peptides were prepared asdescribed in Example 1 and 80 ug of peptide were presented in a totalvolume of 25 ul of sterile HBSS. The peptide solution was applied to theCAM preparation immediately and then again at 24 and 48 hrs. At 72 hoursthe filter paper and surrounding CAM tissue was dissected and viewed asdescribed above.

Results from this assay revealed were similar to those shown in FIGS.9A-9C as described in Example 7E2) where synthetic peptides wereintravenously injected into tumor induced blood vessels. Here, with thecontrol peptide, 62186, the bFGF-induced blood vessels remainedundisturbed as shown in FIG. 9A. In contrast when the cyclic RGDpeptide, 62814, was applied to the filter, the formation of bloodvessels was inhibited leaving the area devoid of new vasculature. Thiseffect was similar in appearance to that shown in FIG. 9B as describedin Example 7E2) below. In addition, also as shown in FIG. 9C forintravenously injected peptides, in areas in which mature blood vesselswere present yet distant from the placement of the growth-factorsaturated filter, no effect was seen with the topical treatment ofsynthetic peptides on these outlying vessels. The inhibitory activity ofthe peptides on angiogenesis thus is limited to the areas ofangiogenesis induced by growth factors and does not effect adjacentpreexisting mature vessels or result in any deleterious cytotoxicity tothe surrounding area.

Similar assays are performed with the other synthetic peptides preparedin Example 1 and listed in Table 1.

3) Treatment with MMP-2 Peptide Fragments

To demonstrate the biological effects of MMP-2 peptide fragments onangiogenesis, CAM assays were performed as described above with theexception that angiogenesis was induced with filter discs saturated for10 minutes with bFGF at a concentration of 1.0 ug/ml in HBS. The discswere then positioned on the CAM in an area that was reduced in thenumber of preexisting vessels. The C-terminal CTMMP-2(410-637) fusionprotein, prepared as described above, or control GST receptor associatedfusion protein (RAP) (1.5 ug in 30 ul of HBSS) was applied thentopically to the filter disc once per day for a total of three days. Atthe end of the incubation period, the embryos were sacrificed and thefilter disc and underlying CAM tissue was resected and analyzed forangiogenesis withya stereo microscope. Angiogenesis was quantified bycounting the number of blood vessels branch points that occur within theconfines of the filter discs. The branched blood vessels are consideredto correspond primarily to new angiogenic sprouting blood vessels.

Quantification was performed in a double blind manner by at least twoindependent observers. The results are expressed as the Angiogenic Indexwhere the angiogenic index is the number of branch points (bFGFstimulated) minus the number of branch points (control unstimulated) perfilter disc. Experiments routinely had 6-10 embryos per condition.

The results of the CAM angiogenesis assay are shown in FIGS. 25A-D, 26and 27. In FIG. 25, a series of photographs divided into four figures,FIGS. 25A-D, illustrate the comparison of angiogenesis inhibited in thepresence of the CTMMP-2 fusion protein (CTMMP-2(410-637)) (FIGS. 25C-D)and not inhibited in the presence of control GST fusion protein (FIGS.25A-B). FIGS. 26 and 27 are bar graphs illustrating the angiogenesisindex of CAM angiogenesis assays with CTMMP-2, the same fusion proteinas above, compared to controls (bFGF only or GST-RAP fusion protein). InFIG. 27, the results of two separate experiments (#1 & #2) usingCTMMP-2(410-637) fusion protein are shown.

These results demonstrated in all three figures indicate that a CTMMP-2fusion protein or polypeptide containing a C-terminal domain of MMP-2 isa useful composition for inhibition of bFGF-mediated angiogenesis byinhibiting α_(v)β₃.

B. Inhibition of Growth Factor-Induced Angiogenesis by IntravenousApplication of Inhibitors

1) Treatment with Monoclonal Antibodies

The effect on growth factor-induced angiogenesis with monoclonalantibodies intravenously injected into the CAM preparation was alsoevaluated for use in this invention.

The preparation of the chick embryo CAMs for intravenous injections wereessentially as described in Example 7A with some modifications. Duringthe candling procedures prominent blood vessels were selected and markswere made on the egg shell to indicate their positions. The holes weredrilled in the shell and the CAMs were dropped and bFGF saturated filterpapers were placed on the CAMs as described above. The windows weresealed with sterile tape and the embryos were replaced in the incubator.Twenty four hours later, a second small window was carefully cut on thelateral side of the egg shell directly over prominent blood vesselsselected previously. The outer egg shell was carefully removed leavingthe embryonic membranes intact. The shell membrane was made transparentwith a small drop of mineral oil (Perkin-Elmer Corp, Norwalk, Conn.)which allowed the blood vessels to be visualized easily. Purifiedsterile mAbs, or synthetic peptides, the latter of which are describedbelow, were inoculated directly into the blood vessels once with a 30gauge needle at a dose of 200 ug of IgG per embryo in a total volume of100 ul of sterile PBS. The windows were sealed with tape and the embryoswere allowed to incubate until 72 hours. The filter disks andsurrounding CAM tissues were analyzed as described before.

To determine the localization of LM609 mAb in CAM tissues or in tumortissues, as shown herein and in the following Examples, that werepreviously inoculated intravenously with LM609, the fixed sections wereblocked with 2.5% BSA in HBSS for 1 hour at room temperature followed bystaining with a 1:250 dilution of goat anti-mouse rhodamine labeledsecondary antibody (Tago). The sections were then analyzed with a Zeissimmunofluorescence compound microscope.

The results of intravenous antibody treatment to the bFGF induced bloodvessel CAM preparation are shown in FIGS. 10A-10C. In FIG. 10A,angiogenesis induced as a result of bFGF treatment is shown. No changeto the presence of bFGF induced vasculature was seen with intravenousexposure to mAb P3G2, an anti-α_(v)β₅ antibody, as shown in FIG. 10B, Incontrast, treatment of the bFGF induced angiogenesis CAM preparationwith LM609, an anti-α_(v)β₃ antibody, resulted in the completeinhibition of growth of new vessels into the filter area as shown inFIG. 10C. The inhibitory effect on angiogenesis is thus resulting fromthe inhibition of α_(v)β₃ receptor activity by the LM609anti-α_(v)β₃-specific antibody. Since the blocking of the α_(v)β₅ doesnot inhibit the formation of neovasculature into the CAMs filter site,α_(v)β₅ thus is not essential as compared to α_(v)β₃ for growth of newvessels.

2) Treatment with Synthetic Peptides

For CAM preparations in which angiogenesis was induced with 1-2 ug/mlbFGF as previously described, synthetic peptides 69601 (control) and66203 (SEQ ID NO 5) were separately intravenously injected into CAMpreparations 18 hours after bFGF induction of angiogenesis. Thepreparations were maintained for an additional 36-40 hours after whichtime the number of branch points were determined as previouslydescribed.

The results are shown in FIG. 28 where peptide 66203 completelyinhibited bFGF-induced angiogenesis in contrast to the absence ofinhibition with the control peptide.

In other assays, peptide 85189 (SEQ ID NO 15) was evaluated forinhibiting bFGF-induced angiogenesis in the CAM assay over a dosagerange of 10 ug/embryo to 300 ug/embryo. The assay was performed aspreviously described. The results are shown in FIG. 29 where the lowesteffective dose was 30 ug with 100 and 300 ug nearly completelyinhibiting angiogenesis.

In still further assays, peptide 85189 was compared to peptides 69601and 66203 for anti-angiogenesis activity. The assay was performed asdescribed above with the exception that 50 ug peptide were used. Theresults, plotted in FIG. 30, showed that peptides 66203 (labeled 203)and 85189 (labeled 189) were effective inhibitors of bFGF-mediatedangiogenesis compared to bFGF-treated (labeled bFGF) and 69601-treated(labeled 601) controls.

The effectiveness of the different salt formulations of peptide 85189was also evaluated in similar bFGF-induced CAM assays. The peptides wereused at 100 ug/embryo. The same peptide sequence in HCl (peptide 85189)and in TFA (peptide 121974) inhibited bFGF-induced angiogenesis with theHCl formulated peptide being slightly more effective than that in TFA(the respective number of branch points for peptide 85189 versus 121974is 30 versus 60). Untreated CAMs, labeled as “no cytokine” hadapproximately half as many branch points as that seen with bFGFtreatment, respectively 70 versus 190. Treatment with control peptide69601 had no effect on inhibiting angiogenesis (230 branch points).

The other synthetic peptides prepared in Example 1 are separatelyintravenously injected into the growth factor induced blood vessels inthe CAM preparation as described above. The effect of the peptides onthe viability of the vessels is similarly assessed.

3) Treatment with MMP-2 Fragments

With the above-described protocol, the effect of MMP-2 fusion proteins,CTMMP-2(2-4), also referred to as CTMMP-2(445-637) and CTMMP-2(10-1),also referred to as CTMMP-2(274-637) was also evaluated. The assay wasperformed as previously described with the exception that 50 ug offusion protein was administered to the bFGF-treated embryos. The effectof fusion protein treatment was assessed at 24 hours, 48 hours and 72hours.

The results are shown for these selected time periods in FIGS. 31A-Lwhere angiogenesis was photographically assessed under assay conditionsof no treatment, bFGF treatment, bFGF treatment followed byCTMMP-2(2-4), labeled as bFGF+MAID (MAID=MMP-2 angiogenesis inhibitingdomain), and bFGF treatment followed by CTMMP-2(10-1), labeled asbFGF+Control. The significant induction of angiogenesis after 48 and 72hours following bFGF treatment was almost completely inhibited only withexposure to CTMMP-2(2-4). The extent of inhibition with CTMMP-2(2-4) wasgreater than that seen with CTMMP-2(10-1) which exhibited some in vivoanti-angiogenesis activity.

The other MMP-2 compositions, whole MMP-2, fragments and fusionproteins, prepared as previously described are also separatelyintravenously injected into the growth factor induced blood vessels inthe CAM preparation as described above. The effect of the peptides onthe viability of the vessels is similarly assessed.

C. Inhibition of Embryonic Angiogenesis by Topical Application

1) Treatment with Monoclonal Antibodies

To determine whether α_(v)β₃ participates in embryonic angiogenesis, theeffect of LM609 on de novo growth of blood vessels on CAMs was examinedin 6 day embryos, a stage marked by active neovascularization asdescribed in Example 5A. The CAM assay was prepared as described inExample 6C with the subsequent topical application of disks saturatedwith mAbs placed on CAMs of 6 day old embryos in the absence ofcytokines. After 3 days, CAMS were resected and photographed. Eachexperiment included 6 embryos per group and was repeated 2 times.

Antibody LM609 (FIG. 11C), but not CSAT (FIG. 11A) or P3G2 (FIG. 11B),prevented vascular growth under these conditions; this indicates thatα_(v)β₃ plays a substantial role in embryonic neovascularization thatwas independent of added growth factors for induction of angiogenesis.

2) Treatment with Synthetic Peptides

The synthetic peptides prepared in Example 1 are separately added to theembryonic CAM preparation prepared above and as described in Example5A2) by either topical application to the CAM or intravenous applicationto blood vessels. The effect of the peptides on the viability of thevessels is similarly assessed.

D. Inhibition of Tumor-Induced Angiogenesis by Topical Application

1) Treatment with Monoclonal Antibodies

In addition to the angiogenesis assays described above where the effectsof anti-α_(v)β₃ antagonists, LM609 and various peptides, on embryonicangiogenesis were evaluated, the role of α_(v)β₃ in tumor-inducedangiogenesis was also investigated. As an inducer, α_(v)β₃-negativehuman M21-L melanoma fragments previously grown and isolated from theCAM of a 17-day chick embryo were used. The fragments were prepared asdescribed in Example 6C.

As described above in Example 7A1), mabs were separately topicallyapplied to the tumor fragments at a concentration of 25 ug in 25 ul ofHBSS and the windows were then sealed with tape. The mAbs were addedagain in the same fashion at 24 hours and 48 hours. At 72 hours, thetumors and surrounding CAM tissues were analyzed as described above inExample 7A1).

As described in Example 6C, tumors were initially derived bytransplanting cultured M21-L cells, which do not to express integrinα_(v)β₃ as described by Felding-Habermann et al., J. Clin. Invest.,89:2018 (1992) onto the CAMs of 10-day old chick embryos. Theseα_(v)β₃-negative fragments induced extensive neovascularization in thepresence of buffer alone, or mAbs CSAT (anti-β₁) or P3G2 (anti-α_(v)β₅).In contrast, mAb LM609 (anti-α_(v)β₃) abolished the infiltration of mostvessels into the tumor mass and surrounding CAM.

In order to quantitate the effect of the mAbs on the tumor-inducedangiogenesis, blood vessels entering the tumor within the focal plane ofthe CAM were counted under a stereo microscope by two observers in adouble-blind fashion. Each data bar presented in FIG. 12 represents themean number of vessels±SE from 12 CAMs in each group representingduplicate experiments.

This quantitative analysis revealed a three-fold reduction in the numberof vessels entering tumors treated with mAb LM609 compared to tumorstreated with buffer or the other mAbs, P3G2 or CSAT (P<0.0001) asdetermined by Wilcoxon Rank Sum Test. The fact that M21-L tumors do notexpress α_(v)β₃ indicates that mAb LM609 inhibits angiogenesis bydirectly affecting blood vessels rather than the tumor cells. Theseresults correspond with the histological distribution of α_(v)β₃ incancer tissue biopsies shown in FIGS. 3A-3D where the distribution ofα_(v)β₃ was limited to the blood vessels in the tumor and not to thetumor cells themselves.

2) Treatment with Synthetic Peptides

The synthetic peptides prepared in Example 1, including MMP-2-derivedpeptides and fusion proteins are topically applied to the tumor-inducedangiogenic CAM assay system as described above. The effect of thepeptides on the viability of the vessels is similarly assessed.

E. Inhibition of Tumor-Induced Anaiogenesis by Intravenous Application

1) Treatment with Monoclonal Antibodies

Tumor-induced blood vessels prepared as described in Example 7E1) werealso treated with mAbs applied by intravenous injection. Tumors wereplaced on the CAMs as described in Example 7D1) and the windows sealedwith tape and 24 hours latter, 200 ug of purified mAbs were inoculatedonce intravenously in chick embryo blood vessels as describedpreviously. The chick embryos were then allowed to incubate for 7 days.The extent of angiogenesis was then observed as described in above. Asdescribed in Example 8 below, after this time period, the tumors wereresected and analyzed by their weight to determine the effect ofantibody exposure on tumor growth or suppression.

2) Treatment with Synthetic Peptides

The effects of peptide exposure to tumor-induced vasculature in the CAMassay system was also assessed. The tumor-CAM preparation was used asdescribed above with the exception that instead of intravenous injectionof a mAb, synthetic peptides prepared as described in Example 1 andExample 7A2) were separately intravenously injected into visible bloodvessels.

The results of CAM assays with the cyclic peptide, 66203 containing theHCl salt, and control peptide, 62186, are shown in FIGS. 9A-9C. In FIG.9A, the treatment with the control peptide did not effect the abundantlarge blood vessels that were induced by the tumor treatment to growinto an area originally devoid of blood vessels of the CAM. In contrastwhen the cyclic RGD peptide, 66203, an antagonist to α_(v)β₃, wasapplied to the filter, the formation of blood vessels was inhibitedleaving the area devoid of new vasculature as shown in FIG. 9B. Theinhibitory effect of the RGD-containing peptide was specific andlocalized as evidenced by an absence of any deleterious effects tovessels located adjacent to the tumor placement. Thus, in FIG. 9C, wheninhibitory peptides are intravenously injected into the CAM assaysystem, no effect was seen on the preexisting mature vessels present inthe CAM in areas adjacent yet distant from the placement of the tumor.The preexisting vessels in this location were not affected by theinhibitory peptide that flowed within those vessels although thegeneration of new vessels from these preexisting vessels into the tumormass was inhibited. Thus, synthetic peptides including 66203 and 62184,previously shown in ligand-receptor assays in Example 4 to beantagonists of α_(v)β₃, have now been demonstrated to inhibitangiogenesis that is limited to vessels undergoing development and notto mature preexisting vessels. In addition, the intravenous infusion ofpeptides does not result in any deleterious cytotoxicity to thesurrounding area as evidence by the intact vasculature in FIG. 9C.

Similar assays are performed with the other synthetic peptides preparedin Example 1 and listed in Table 1 along with the MMP-2 compositions ofthis invention.

3) Treatment with MMP-2 Fragments

A CS-1 tumor (β₃-negative) was prepared in a CAM as described above.After 24 hours of tumor growth, a composition of MMP-2 fragment,designated CTMMP-2(2-4) and prepared as described in Example 4A, wasadministered intraveneously at 50 ug fragment in 100 ul of PBS. After 6days, the tumor was evaluated for mass. Tumors treated with CTMMP-2(2-4)were reduced in growth rate by about 50% when compared to the growthrate of control tumors treated with CTMMP-2(10-1) or with PBS control.Thus, the α_(v)β₃ antagonist inhibited tumor growth.

8. Inhibition of Tumor Tissue Growth with α_(v)β₃ Antagonists asMeasured in the CAM Assay

As described in Example 7E1), in addition to visually assessing theeffect of anti-α_(v)β₃ antagonists on growth factor or tumor inducedangiogenesis, the effect of the antagonists was also assessed bymeasuring any changes to the tumor mass following exposure. For thisanalysis, the tumor-induced angiogenesis CAM assay system was preparedas described in Example 6C and 7D. At the end of the 7 day incubationperiod, the resulting tumors were resected from the CAMs and trimmedfree of any residual CAM tissue, washed with 1 ml of phosphate buffersaline and wet weights were determined for each tumor.

In addition, preparation of the tumor for microscopic histologicalanalysis included fixing representative examples of tumors in BulinsFixative for 8 hours and embedding in paraffin. Serial sections were cutand stained with hematoxylin and eosin (H&E) for microscopic analysis.Gladson, et al., J. Clin. Invest., 88:1924 (1991). Sections werephotographed with an Olympus compound microscope at 250×.

A. Topical Application

The results of typical human melanoma tumor (M21L) weights resultingfrom topical application of control buffer (HBSS), P3G2 (anti-α_(v)β₅)or LM609 (anti-α_(v)β₃) are listed in Table 4. A number of embryos wereevaluated for each treatment with the average tumor weight in milligrams(mg) from each being calculated along with the SE of the mean as shownat the bottom of the table.

TABLE 4 Embryo No. mAb Treatment Tumor Weight (mg) 1 HBSS 108 2 152 3216 4 270 5 109 6 174 1 P3G2 134 2 144 3 408 4 157 5 198 6 102 7 124 899 1 LM609 24 2 135 3 17 4 27 5 35 6 68 7 48 8 59 mAb Treatment AverageTumor Weight (mg) HBSS control 172 ± 26 P3G2 171 ± 36 LM609   52 ± 13

Exposure of a α_(v)β₃-negative human melanoma tumor mass in the CAMassay system to LM609 caused the decrease of the untreated average tumorweight of 172 mg±26 to 52 mg±13. The P3G2 antibody had no effect on thetumor mass. Thus, the blocking of the α_(v)β₃ receptor by the topicalapplication of α_(v)β₃-specific LM609 antibody resulted in a regressionof tumor mass along with an inhibition of angiogenesis as shown in thepreceding Examples. The measured diameter of the tumor mass resultingfrom exposure to P3G2 was approximately 8 millimeters to 1 centimeter onaverage. In contrast, the LM609-treated tumors were on average 2 to 3millimeters in diameter.

Frozen sections of these tumors revealed an intact tumorcytoarchitecture for the tumor exposed to P3G2 in contrast to a lack ororganized cellular structure in the tumor exposed to LM609. α_(v)β₃receptor activity is therefore essential for an α_(v)β₃ negative tumorto maintain its mass nourished by development of α_(v)β₃-expressingneovasculature. The blocking of α_(v)β₃ with the α_(v)β₃ antagonists ofthis invention results in the inhibition of angiogenesis into the tumorultimately resulting in the diminution of tumor mass.

B. Intravenous Application

The results of typical carcinoma tumor (UCLAP-3) weights resulting fromintravenous application of control buffer (PBS, phosphate bufferedsaline), CSAT (anti-β₁ ) or LM609 (anti-α_(v)β₃) are listed in Table 5.A number of embryos were evaluated for each treatment with the averagetumor weight from each being calculated along with the SE of the mean asshown at the bottom of the table.

TABLE 5 Embryo No. mAb Treatment Tumor Weight (mg) 1 PBS 101 2 80 3 67 490 1 CSAT 151 2 92 3 168 4 61 5 70 1 LM609 16 2 54 3 30 4 20 5 37 6 39 712 mAb Treatment Average Tumor Weight (mg) PBS control 85 ± 7 CSAT 108 ±22 LM609 30 ± 6

Exposure of a α_(v)β₃-negative human carcinoma tumor mass in the CAMassay system to LM609 caused the decrease of the untreated average tumorweight of 85 mg±7 to 30 mg±6. The CSAT antibody did not significantlyeffect the weight of the tumor mass. Thus, the blocking of the α_(v)β₃receptor by the intravenous application of α_(v)β₃-specific LM609antibody resulted in a regression of a carcinoma as it did for themelanoma tumor mass above along with an inhibition of angiogenesis asshown in the preceding Examples. In addition, human melanoma tumorgrowth was similarly inhibited by intravenous injection of LM609.

9. Regression of Tumor Tissue Growth with α_(v)β₃ Antaaonists asMeasured in the CAM Assay

To further assess the effects of α_(v)β₃ antagonists on tumor growth andsurvival, fragments of human melanoma and fragments of carcinomas of thelung, pancreas, and larynx were placed on CAMS of 10-day old embryos asdescribed in Example 5A.

A. Intravenous Application

1) Treatment with Monoclonal Antibodies

a. Treatment with LM609 (Anti-α_(v)β₃) and CSAT (Anti-B₁)

Twenty four hours after implantation of CAM with carcinoma fragments ofα_(v)β₃-negative human melanoma M21-L, pancreatic carcinoma FG, humanlung carcinoma UCLAP-3, or human laryngeal carcinoma HEp3, embryos wereinjected intravenously with PBS alone or a single dose (300 ug/100 ul)of either mAb LM609 (anti-α_(v)β₃) or CSAT (anti-β₁). Tumors wereallowed to propagate for six additional days. At the end of theincubation period the tumors were carefully resected and trimmed free ofsurrounding CAM tissue. Tumor resections were performed by twoindependent investigators removing only the easily definable solid tumormass. The tumors had well defined margins, thus the thinsemi-transparent membrane (CAM) which is readily distinguishable fromthe solid tumor mass was removed without disturbing the tumor massitself. The resected tumors were weighed and examined morphologicallyand histologically.

As shown in FIG. 13, wet tumor weights at the end of 7 days weredetermined and compared to initial tumor weights before treatments. Eachbar represents the mean±S.E. of 5-10 tumors per group. mAb LM609inhibited tumor growth significantly (p<0.001) as compared to controlsin all tumors tested. Tumors treated with PBS or CSAT proliferated inall cases. In contrast, mAb LM609 not only prevented the growth of thesetumors but induced extensive regression in most cases. Importantly,these tumor cells do not express integrin α_(v)β₃ demonstrating that theinhibition of growth was due to the anti-angiogenic effects of thisantibody on neovasculature rather than upon the tumor cells directly.

b. Treatment with LM609 (Anti-α_(v)β₃) and P3G2 (Anti-α_(v)β₅)

Human M21-L melanoma tumor fragments (50 mg) were implanted on the CAMsof 10 day old embryos as described in Example 5A. Twenty four hourslater, embryos were injected intravenously with PBS alone or a singledose (300 ug/100 ul) of either mAb LM609 (anti-α_(v)β₃) or P3G2(anti-α_(v)β₅). Tumors were allowed to propagate as described in Example9A1) a above and were examined morphologically and histologically asherein described.

Representative examples of M21-L tumors treated with mabs P3G2(anti-α_(v)β₅) or LM609 (anti-α_(v)β₃) were examined morphologically.The P3G2-treated tumors were large (8 mm in diameter) and wellvascularized whereas those treated with mAb LM609 were much smaller (3mm in diameter) and lacked detectable blood vessels.

The tumors were further examined by the preparation of histologicalsections and staining with hematoxylin and eosin as described in Example9A1)a. As shown in FIG. 14 (upper panel), tumors treated with mAb P3G2(anti-α_(v)β₅) showed numerous viable and actively dividing tumor cellsas indicated by mitotic figures (arrowheads) as well as by multipleblood vessels (arrows) throughout the tumor stroma. In contrast, few ifany viable tumor cells or blood vessels were detected in tumors treatedwith mAb LM609 (anti-α_(v)β₃) (FIG. 14, lower panel). These resultsdemonstrate that antagonists of integrin α_(v)β₃ inhibit tumor-inducedangiogenesis leading to the growth arrest and regression of a variety ofhuman tumors in vivo. It is important to point out that embryos examinedafter seven days of tumor growth (embryonic day 17) appeared normal upongross examination whether or not they were treated with an α_(v)β₃antagonist. These findings indicate that antagonists of this integrinappear non-toxic to the developing embryos.

2) Treatment with Synthetic Peptides

Human M21-L melanoma tumor fragments (50 mg) were implanted on the CAMsof 10 day oldembryos as described in Example 5A. Twenty four hourslater, embryos received a single intravenous injection of 300 ug/100 ulof either the cyclo-RADfV (69601) and or cyclo-RGDfV (66203). After atotal of 72 hours, tumors were removed, examined morphologically, andphotographed with a stereo microscope as described in Example 9A1).

The panels shown in FIGS. 15A through 15E correspond as follows: FIG.15A, duplicate samples treated with cyclo-RADfV peptide (69601); FIG.15B, duplicate samples treated with cyclo-RGDfV peptide (66203); FIG.15C, adjacent CAM tissue taken from the same embryos treated withcyclo-RGDfV peptide (66203) and FIGS. 15D and 15E, high magnification(13×) of peptide treated tumors. FIG. 15D depicts normal blood vesselsfrom control peptide (69601) treated tumor. FIG. 15E depicts examples ofdisrupted blood vessels from cyclo-RGDfV peptide (66203) treated tumors(arrows).

The results illustrate that only peptide 66203 in contrast to controlpeptide 69601 inhibited vessel formation, and further that vessels inthe CAM tissue adjacent to the tumor were not affected.

Additional tumor regression assays were performed with theα_(v)β₃-reactive peptide 85189 (SEQ ID NO 15) against 69601 as acontrol. The assays were performed as described above with the exceptionthat 100 ug of peptide was intravenously injected into the CAM at 18hourst postimplantation. After 48 hours more, the tumors were thenresected and wet weights were obtained.

FIGS. 32, 33 and 34 respectively show the reduction in tumor weight forUCLAP-3, M21-L and FgM tumors following intravenous exposure to peptide85189 in contrast to the lack of effect with either PBS or peptide69601.

10. Regression of Tumor Tissue Growth with α_(v)β₃ Antagonists asMeasured by in Vivo Rabbit Eye Model Assay

The effect of anti-α_(v)β₃ antagonists on growth factor-inducedangiogenesis can be observed in naturally transparent structures asexemplified by the cornea of the eye. New blood vessels grow from therim of the cornea, which has a rich blood supply, toward the center ofthe cornea, which normally does not have a blood supply. Stimulators ofangiogenesis, such as bFGF, when applied to the cornea induce the growthof new blood vessels from the rim of the cornea. Antagonists ofangiogenesis, applied to the cornea, inhibit the growth of new bloodvessels from the rim of the cornea. Thus, the cornea undergoesangiogenesis through an invasion of endothelial cells from the rim ofthe cornea into the tough collagen-packed corneal tissue which is easilyvisible. The rabbit eye model assay therefore provides an in vivo modelfor the direct observation of stimulation and inhibition of angiogenesisfollowing the implantation of compounds directly into the cornea of theeye.

A. In Vivo Rabbit Eye Model Assay

1) Angiogenesis Induced by Growth Factors

Angiogenesis was induced in the in vivo rabbit eye model assay with thegrowth factor bFGF and is described in the following sections.

a. Preparation of Hydron Pellets Containing Growth Factor and MonoclonalAntibodies

Hydron polymer pellets containing growth factor and mAbs were preparedas described by D'Amato, et al., Proc. Natl. Acad. Sci.. USA,91:4082-4085 (1994). The individual pellets contained 650 ng of thegrowth factor bFGF bound to sucralfate (Carafet, Marion Merrell DowCorporation) to stabilize the bFGF and ensure its slow release into thesurrounding tissue. In addition, hydron pellets were prepared whichcontained either 40 ug of the mAb LM609 (anti-α_(v)β₃) or mAb P1F6(anti-α_(v)β₅) in PBS. The pellets were cast in specially preparedTeflon pegs that have a 2.5 mm core drilled into their surfaces.Approximately 12 ul of casting material was placed into each peg andpolymerized overnight in a sterile hood. Pellets were then sterilized byultraviolet irradiation.

b. Treatment with Monoclonal Antibodies

Each experiment consisted of three rabbits in which one eye received apellet comprising bFGF and LM609 and the other eye received a pelletcomprising bFGF and a mouse mAb P1F6 (anti-α_(v)β₅). The use of pairedeye testing to compare LM609 (anti-α_(v)β₃) to other mAb and PBScontrols provides a means for rigorous testing to demonstratesignificant differences between the mAbs tested.

The P1F6 mAb immunoreacts with the integrin α_(v)β₅ which is found onthe surface of vascular endothelial cells but is presumably not involvedin angiogenesis. To determine whether the mAb P1F6 was involved inangiogenesis, pellets containing only this mAb were prepared and assayedas described below to confirm that the mAb did not induce angiogenesis.

All of the mAbs tested were purified from ascites fluid using Protein-ASepharose CL-4B affinity column chromatography according to well-knownmethods. The eluted immunoglobulin was then dialyzed against PBS andtreated with Detoxi-gel (Pierce Chemicals) to remove endotoxin.Endotoxin has been shown to be a potent angiogenic and inflammatorystimulant. mAbs were therefore tested for the presence of endotoxin withthe Chromogenic Limulus Amebocyte Lysate Assay (Bio-Whittaker) and onlythose mAbs without detectable endotoxin were used in the rabbit eyemodel assay.

A hydron pellet comprising bFGF and mAb LM609 (anti-α_(v)β₃) or P1F6(anti-α_(v)β₅) was inserted into a corneal pocket formed in the eye ofrabbits. The hydron pellet also contained sucralfate to stabilize thebFGF during the assay. Individual pellets were implanted into surgicallycreated “pockets” formed in the mid-stroma of the cornea of rabbits. Thesurgical procedure was done under sterile technique using a Wild modelM691 operating microscope equipped with a beamsplitter to which wasmounted a camera for photographically recording individual corneas. A 3mm by 5 mm “pocket” was created in the corneal stroma by making a 3 mmincision to half the corneal thickness with a 69 Beaver blade. Thestroma was dissected peripherally using an iris spatula and the pelletwas implanted with its peripheral margin 2 mm from the limbus.

During the following 14 days, bFGF and mAb diffused from the implantedpellet into the surrounding tissue and thereby effected angiogenesisfrom the rim of the cornea.

Representative results of each treatment are depicted in FIGS. 16Athrough 16E. The amount of vessels present are quantitated and describedin terms of clock hours which are defined as follows. The eye is dividedinto 12 equal sections in the same manner as a clock is divided intohours. “One clock hour of vessels” refers to that amount of vesselswhich fills an area of the eye equivalent to one hour on a clock. Thefive rabbits which received only bFGF exhibited florid angiogenesis inwhich new blood vessels had grown from the rim of the cornea toward thecenter of the cornea, which normally does not have blood vessels. One ofthese rabbits had only 1 clock hour of vessels to the pellet. Two of therabbits which received both bFGF and mAb LM609 had absolutely nodetectable angiogenesis up to 14 days following surgery. One of theserabbits had 3 foci of hemorrhagic and budding vessels by day 14. Two ofthe rabbits which received bFGF and mAb P3G2 (anti-α_(v)β₅) showedextensive vascularization in which new blood vessels had grown from therim of the cornea into the cornea. One of these rabbits had only 1 to 2hours of vessels to the pellet.

As evidenced in the rabbit eye model assay, no angiogenic effect wasobserved on normal paralimbal vessels in the presence of the growthfactor bFGF in rabbits which received mAb LM609 (anti-α_(v)β₃). Incontrast, angiogenesis was observed on paralimbal vessels in thepresence of the growth factor bFGF in rabbits which received the mAbP3G2 (anti-α_(v)β₅). The complete inhibition of corneal angiogenesis bymAb LM609 is substantially greater than any previously reportedanti-angiogenic reagent.

c. Treatment with Polypeptides

Each experiment consisted of eight rabbits in which one eye received apellet comprising 100 nanograms (ng) bFGF and the other eye received apellet comprising 1 microgram (ug) VEGF. The pellets were inserted intothe corneal pocket as described above, and the cytokines subsequentlystimulated the growth of new blood vessels into the cornea. Peptideswere administered subcutaneously (s.q.) in 1 ml PBS at an initial dosageof 50 ug per kg rabbit the day of pellet insertion, and daily s.q.dosages were given at 20 ug/kg thereafter. After 7 days, the cornea wereevaluated as described above.

Rabbits receiving control peptide 69601 showed substantial corneal bloodvessel growth at 7 days, in both vFGF and VEGF stimulated eyes. Rabbitsreceiving peptide 85189 showed less than 50% of the amount of cornealblood vessel growth compared to controls in vFGF-stimulated eyes andnearly 100% inhibition in VEGF-stimulated eyes.

11. In Vivo Regression of Tumor Tissue Growth with α_(v)β₃ Antagonistsas Measured by Chimeric Mouse:Human Assay

An in vivo chimeric mouse:human model was generated by replacing aportion of skin from a SCID mouse with human neonatal foreskin (FIG.17). After the skin graft was established, the human foreskin wasinoculated with carcinoma cells. After a measurable tumor wasestablished, either mAb LM609 (anti-α_(v)β₃) or PBS was injected intothe mouse tail vein. Following a 2-3 week period, the tumor was excisedand analyzed by weight and histology.

A. In Vivo Chimeric Mouse:Human Assay

The in vivo chimeric mouse:human model is prepared essentially asdescribed in Yan, et al., J. Clin. Invest., 91:986-996 (1993). Briefly,a 2 cm² square area of skin was surgically removed from a SCID mouse(6-8 weeks of age) and replaced with a human foreskin. The mouse wasanesthetized and the hair removed from a 5 cm² area on each side of thelateral abdominal region by shaving. Two circular graft beds of 2 cm²were prepared by removing the full thickness of skin down to the fascia.Full thickness human skin grafts of the same size derived from humanneonatal foreskin were placed onto the wound beds and sutured intoplace. The graft was covered with a Band-Aid which was sutured to theskin. Micropore cloth tape was also applied to cover the wound.

The M21-L human melanoma cell line or MDA 23.1 breast carcinoma cellline (ATCC HTB 26; α_(v)β₃ negative by immunoreactivity of tissuesections with mAb LM609), were used to form the solid human tumors onthe human skin grafts on the SCID mice. A single cell suspension of5×10⁶ M21-L or MDA 23.1 cells was injected intradermally into the humanskin graft. The mice were then observed for 2 to 4 weeks to allow growthof measurable human tumors.

B. Intravenous Application

1) Treatment With Monoclonal Antibodies

Following the growth of measurable tumors, SCID mice, which had beeninjected with M21L tumor cells, were injected intravenously into thetail vein with 250 μg of either the mAb LM609 (anti-α_(v)β₃) or PBStwice a week for 2 to 3 weeks. After this time, the tumors were resectedfrom the skin and trimmed free of surrounding tissue. Several mice wereevaluated for each treatment with the average tumor weight from eachtreatment being calculated and shown at the bottom of Table 6.

TABLE 6 M21L Tumor Number Treatment Tumor Weight (mg) 1 PBS 158 2 192 3216 4 227 5 LM609 195 6 42 7 82 8 48 9 37 10  100 11  172 TreatmentAverage Tumor Weight (mg) PBS 198 LM609 113

Exposure of the M21L α_(v)β₃-negative human carcinoma tumor mass in themouse:human chimeric assay system to LM609 (anti-α_(v)β₃) caused thedecrease from the PBS treated average tumor weight of 198 mg to 113 mg.

Representative examples of M21L tumors treated with the mAb LM609(anti-α_(v)β₃) and PBS were examined morphologically. The PBS-treatedtumors were large (8 to 10 mm in diameter) and well vascularized whereasthose treated with mAb LM609 (anti-α_(v)β₃) were much smaller (3 to 4 mmin diameter) and lacked detectable blood vessels.

In other experiments with M21-L melanoma tumor cells in the mouse:humanchimeric assay system, the response with mAB LM609 was compared with theresponse obtained with the synthetic peptide 85189 (SEQ ID NO 15) ascompared to control synthetic peptide 69601 (SEQ ID NO 6). The assayswere performed as described above. The results, shown in FIG. 35,demonstrate that the synthetic peptide 85189 reduced tumor volume tobelow 25 mm³ as compared to control peptide where the tumor volume wasapproximately 360 mm³. The mAB LM609 also significantly reduced tumorvolume to approximately 60 mm³.

Tumors formed in skin grafts which had been injected with MDA 23.1 cellswere detectable and measurable. Morphological examination of theestablished tumors revealed that neovascularization from the graftedhuman tissue into the MDA 23.1 tumor cells had occurred.

Thus, blocking of the α_(v)β₃ receptor by the intravenous application ofα_(v)β₃-specific LM609 antibody and peptides resulted in a regression ofa carcinoma in this model system in the same manner as the CAM andrabbit eye model systems as described in Examples 9 and 10,respectively.

2) Treatment with Synthetic Peptides

In a procedure similar to that described above for monoclonalantibodies, peptide antagonists of α_(v)β₃ were injected intravenouslyinto the tail vein of SCID mice having measurable M21-L tumors. In apreliminary analysis, a dose response curve was performed for peptides69601 (control) and 85189 (test) injected over a concentration range of10 to 250 ug/ml. The mean volume and weight of resected tumors followingtreatment were determined with the results respectively shown in FIGS.36A and 36B. Peptide 85189 was effective at inhibiting M21-L tumorgrowth over the concentration range tested compared to treatment withcontrol peptide with the most effective dosage being 250 ug/ml.

For analyzing peptide 85189 treatment effectiveness over a time course,two treatment regimens were evaluated in the same SCID tumor model. Inone assay, treatment with either peptide 85189 or 69601 was initiated onday 6, with day 0 being the day of M21-L tumor injection of 3×10⁶ cellssubcutaneously into mouse skin, with intraperitoneal injections of 250ug/ml peptide 85189 or control 69601 every other day until day 29. Theother assay was identically performed with the exception that treatmentwas initiated on day 20. At the end of the assays, the tumors wereresected and the mean tumor volume in mm³ was determined. The data wasplotted as this value +/− the standard error of the mean.

The results of these assays, respectively shown in FIGS. 37A and 37B,indicate that peptide 85189 but not 69601 inhibited tumor growth atvarious days after treatment was initiated, depending on the particulartreatment regimen. Thus, peptide 85189 is an effective α_(v)β₃antagonist of both angiogenesis and thus tumor growth.

12. Stimulation of Vascular Cells to Enter the Cell Cycle and UndergoApoptosis in the Presence of Antagonists of Intearin α_(v)β₃ as Measuredin the CAM Assay

The angiogenic process clearly depends on the capacity of cytokines suchas bFGF and VEGF to stimulate vascular cell proliferation. Mignatti etal., J. Cell. Biochem., 471:201 (1991); Takeshita et al., J. Clin.Invest., 93:662 (1994); and Koyama et al., J. Cell. Physiol., 158:1(1994). However, it is also apparent that signaling events may regulatethe differentiation of these vascular cells into mature blood vessels.Thus, it is conceivable that interfering with signals related to eithergrowth or differentiation of vascular cells undergoing new growth orangiogenesis may result in the perturbation of angiogenesis.

Integrin ligation events have been shown to participate in both cellproliferation as well as apoptosis or programmed cell death in vitro.Schwartz, Cancer Res., 51:1503 (1993); Meredith et al., Mol. Biol.Cell., 4:953 (1993); Frisch et al., J. Cell Biol., 124:619 (1994); andRuoslahti et al., Cell, 77:477 (1994). Close examination of the effectsof α_(v)β₃ antagonists on angiogenesis reveals the presence ofdiscontinuous and disrupted tumor-associated blood vessels. Therefore,it is possible that the loss of blood vessel continuity may be due toselective necrosis or apoptosis of vascular cells.

To explore this possibility, CAMs were examined after induction ofangiogenesis with the growth factor bFGF and treatment with the mAb andcyclic peptides of this invention.

A. Treatment with Monoclonal Antibodies

Apoptosis can be detected by a variety of methods which include directexamination of DNA isolated from tissue to detect fragmentation of theDNA and the detection of 3′OH in intact tissue with an antibody thatspecifically detects free 3′OH groups of fragmented DNA.

1) Analysis of DNA Fragmentation

Angiogenesis was induced by placing filter disks saturated with bFGF onthe CAMs of 10-day old embryos as described in Examples 6A.Immunohistological analysis of CAMs with LM609 (anti-α_(v)β₃) revealedpeak expression of α_(v)β₃ on blood vessels 12 to 24 hours afterinitiation of angiogenesis with bFGF. Thus, 24 hours after stimulationwith bFGF, embryos were inoculated intravenously with 100 μl of PBSalone or PBS containing 300 μg of either mAb CSAT (anti-β₁) or LM609(anti-α_(v)β₃).

DNA fragmentation was detected by resecting the CAM tissue directlybelow bFGF saturated filter disks 24 or 48 hours after intravenousinoculations with mAb LM609 (anti-α_(v)β₃), CSAT (anti-β₁), or PBS.Resected CAM tissues were washed three times with sterile PBS and finelyminced, resuspended in 0.25% bacterial collagenase (WorthingtonBiochemical; Freehold, N.J.) and incubated for 90 minutes at 37 C. withoccasional vortexing. DNA was extracted from equal numbers of CAM cellsfrom single cell suspension as previously described. Bissonette, et al.,Nature, 359:552 (1992). Briefly, equal numbers of CAM cells were lysedin 10 mM Tris-HCl, pH 8.0, 10 mM EDTA in 0.5% (v/v) Triton X-100 (Sigma,St. Louis, Mo.). Cell lysates were centrifuged at 16,000×g for 15minutes at 4 C. to separate soluble fragmented DNA from the intactchromatin pellet. Fragmented DNA was washed, precipitated, and analyzedon a 1.2% (w/v) agarose gel.

Soluble fragmented DNA was isolated from an equal number of CAM cellsfrom each treatment, separated electrophoretically on an agarose gel,and visualized by staining with ethidium bromide. No difference wasdetected in the relative amount of DNA fragmentation resulting from thethree different treatments 24 hours after treatment. However, by 48hours following treatment with mAb LM609 (anti-α_(v)β₃), a significantincrease in DNA fragmentation was observed when compared to embryostreated with either mAb CSAT (anti-β₁) or PBS alone.

2) Stimulation of Vascular Cells to Enter the Cell Cycle

To experimentally examine the role of α_(v)β₃ in these processes, cellsderived from CAMs treated with or without bFGF were stained withpropidium iodide and immunoreacted with mAb LM609 (anti-α_(v)β₃).

CAMs isolated from embryos 24 and 48 hours after treatment with mAbLM609 (anti-α_(v)β₃), CSAT (anti-β₁), or PBS were dissociated intosingle cell suspensions by incubation with bacterial collagenase asdescribed above. Single cells were then permeabilized and stained withApop Tag Insitu Detection Kit according to the manufacturer'sinstructions (Oncor, Gaithersburg, Md.). Apop Tag is an antibody thatspecifically detects free 3′OH groups of fragmented DNA. Detection ofsuch free 3′OH groups is an established method for the detection ofapoptotic cells. Gavrieli et al., J. Cell Biol., 119:493 (1992).

Apop Tag stained cells were then rinsed in 0.1% (v/v) Triton X-100 inPBS and resuspended in FACS buffer containing 0.5% (w/v) BSA, 0.02%(w/v) sodium azide and 200 ug/ml RNase A in PBS. Cells were incubatedfor 1.5 hrs, washed, and analyzed by fluorescence activated cellsorting. Cell fluorescence was measured using a FACScan flow cytometerand data analyzed as described below.

Cell fluorescence was measured with a FACScan flow cytometer (BectonDickinson, Mountain View, Calif.). Side scatter (SSC) and forwardscatter (FSC) were determined simultaneously and all data were collectedwith a Hewlet Packard (HP9000) computer equipped with FACScan researchsoftware (Becton Dickinson, Mountain View, Calif.). The data wereanalyzed with P.C Lysis version I software (Becton Dickinson, MountainView, Calif.). Negative control gates were set by using cell suspensionswithout the addition of primary antibodies from the Apop Tag kit.Identical gating was applied to both cell populations resulting in theanalysis of approximately 8,000 cells per different cell treatment.

The percent of single cells derived from mAb treated CAMs and stainedwith Apop Tag as determined by FACS analysis is shown in FIG. 18. Theblack bar represents cells from embryos treated 24 hours prior toanalysis. The stippled bar represents cells from embryos treated 48hours prior to analysis. Each bar represents the mean±S.E. of threereplicates.

As shown in FIG. 18, CAMs treated two days prior with mAb LM609(anti-α_(v)β₃) showed a 3 to 4-fold increase in Apop Tag staining ascompared to CAMs treated with either PBS alone or CSAT (anti-β₁).

B. Treatment with Synthetic Peptides

CAM assays with growth factor-induced angiogenesis, as described inExample 6A, were also performed with the synthetic peptides of thisinvention to determine the effect of cyclic peptides on apoptosis. Thepeptides cyclo-RGDfV (66203) and cyclo-RADfV (69601) were prepared asdescribed in Example 1. The peptide solutions or PBS were injected intothe CAM preparation at a concentration of 300 ug/ml. At 24 and 48 hours,the filter paper and surrounding CAM tissue was dissected and stainedwith the Apop Tag to detect apoptosis as described above in Example12A2).

As shown in FIG. 18, CAMs treated two days prior with peptide 69203(cyclo-RGDfV) showed a 3 to 4-fold increase in Apop Tag staining ascompared to CAMs treated with either PBS alone or control cyclic peptide69601 (cyclo-RADfV).

C. Effect of Treatment with Monoclonal Antibodies on Apoptosis and CellCycle

Single cell suspensions were also examined for the number of copies ofchromosomal DNA by staining with propidium iodide to determine theeffect of treatment with monoclonal antibodies on the cell cycle and forapoptosis by staining with the Apop Tag.

Single cell suspensions of CAMS treated 24 or 48 hours prior with mAbLM609 (anti-α_(v)β₃) or CSAT (anti-β₁) or PBS were prepared as describedin Example 12A1).

For staining of cells with the Apop Tag, cell suspensions were washedthree times with buffer containing 2.5% (w/v) BSA and 0.25% (w/v) sodiumazide in PBS. Cells were then fixed in 1% (w/v) paraformaldehyde in PBSfor 15 minutes followed by three washes as described above. To preventnonspecific binding, single cell suspensions were blocked with 5 (w/v)BSA in PBS overnight at 4 C. Cells were then washed as before, stainedwith Apop Tag, and cell fluorescence measured with a FACScan asdescribed above in Example 12A.

Cells from each experimental condition were stained with propidiumiodide (Sigma, St. Louis, Mo.) at 10 ug/ml in PBS for 1 hour, washed twotimes with PES, and analyzed for nuclear characteristics typical ofapoptosis, including chromatin condensation and segmentation. Thepercentage of apoptotic cells were estimated by morphological analysisof cells from at least 10 to 15 randomly selected microscopic fields.

The combined results of single cell suspensions of CAMs from embryostreated with either CSAT (anti-β₁) or LM609 (anti-α_(v)β₃), stained withApop Tag and propidium iodide, and analyzed by FACS are given in FIG.19. The Y axis represents Apop Tag staining (apoptosis), the X axisrepresents propidium iodide staining (DNA content). The horizontal linerepresents the negative gate for Apop Tag staining. The left and rightpanels indicate CAM cells from CSAT and LM609 treated embryos,respectively. Cell cycle analysis was performed by analysis ofapproximately 8,000 events per condition and data represented in acontour plot.

Samples of single cells stained with the DNA dye propidium iodiderevealed that 25-30%i of the LM609 (anti-α_(v)β₃) treated CAM cells 48hours after treatment showed evidence of nuclear condensation and/orsegmentation. These processes are characteristic of cells undergoingapoptosis. This is in contrast to CAMs treated with CSAT (anti-β₁) where90-95w of the cells showed normal nuclear staining.

As shown in FIG. 19, consistent with the induction of apoptosis byLM609, a significant number of cells in a peak containing less than onecopy of DNA was observed (AO). This peak has been previously shown torepresent fragmented DNA in late stage apoptotic cells. Telford et al.,Cytometry, 13:137 (1992). Furthermore, these AO cells readily stain withApop Tag confirming the ability of this reagent to detect apoptoticcells. However, in addition to the staining of cells in AO, asignificant number of cells containing greater than one copy of DNA alsostained with Apop Tag (FIG. 19). These results demonstrate the LM609 hasthe ability to promote apoptosis among vascular cells that had alreadyentered the cell cycle. In contrast, cells derived from control CAMswhich had entered the cell cycle showed minimal Apop Tag stainingconsistent with the few apoptotic cells detected in control treatedCAMs.

Among those cells in the bFGF stimulated CAMs that had entered the cellcycle (S and G2/M phase), 70% showed positive staining with LM609(anti-α_(v)β₃). This is compared to 10% LM609 staining observed amongcycling cells from non-bFGF treated CAMs. These findings indicate thatafter bFGF stimulation, the majority of the α_(v)β₃-bearing cells showactive proliferation.

Taken together these findings indicate that intravenous injection of mAbLM609 or cyclic peptide antagonist of α_(v)β₃ promote apoptosis withinthe chick CAM following induction of angiogenesis.

CAMs were also examined histologically for expression of α_(v)β₃ byimmunoreactivity with LM609 and for cells which were undergoingapoptosis by immunoreactivity with Apop Tag. CAM sections resected fromembryos treated 48 hours prior with LM609 (anti-α_(v)β₃), CSAT(anti-β₁), or PBS prepared in Example 5A were washed, embedded in OTC(Baxter) and snap frozen in liquid nitrogen. Six micron sections of CAMtissues were cut, fixed in acetone for 30 seconds, and stored at −70 C.until use. Tissue sections were prepared for staining by a brief rinsein 70% (v/v) ethanol (ETOH) followed by washing three times in PBS.Next, sections were blocked with 5% (w/v) BSA in PBS for 2 hours,followed by incubation with 10 ug/ml of mAb LM609 for 2 hours. Thesections were then washed and incubated witha 1:50 dilution of rhodamineconjugated goat anti-mouse IgG (Fisher Scientific, Pittsburg, Pa.) for 2hours. Finally, the same sections were washed and stained with the ApopTag as described in Example 12A2). The stained tissue sections weremounted and analyzed by confocal immunofluorescent microscopy.

In FIG. 20, panels A through C represent CAM tissue from CSAT (anti-β₁)treated embryos and panels D through F represent CAM tissue from LM609(anti-α_(v)β₃) treated embryos. Panels A and D depict tissues stainedwith Apop Tag and visualized by fluorescence (FITC) superimposed on aD.I.C. image. Panels B and E depict the same tissues stained with mAbLM609 (anti-α_(v)β₃) and visualized by fluorescence (rhodamine). PanelsC and F represent merged images of the same tissues stained with bothApop Tag and LM609 where yellow staining represents colocalization. Thebar represents 15 and 50 μm in the left and right panels, respectively.

As shown in FIGS. 20(A-C), after intravenous injection of CSAT or PBScontrol, staining with Apop Tag appeared minimal and random, indicatinga minimal level of apoptosis within the tissue. In contrast, CAMs fromembryos previously treated with LM609 or cyclic peptide 203 showed amajority of the vessels staining intensely with Apop Tag while minimalreactivity was observed among surrounding nonvascular cells (FIGS.20D-F). Furthermore, when both Apop Tag and LM609 were used to stainthese tissues (19C and 19F) significant co-localization was onlyobserved between these markers in CAMs derived from embryos treated withα_(v)β₃ antagonists (FIG. 20F). These findings demonstrate that afterinduction of angiogenesis in vivo, inhibitors of integrin α_(v)β₃selectively promote apoptosis of α_(v)β₃-bearing blood vessels.

While angiogenesis is a complex process involving many molecular andcell biological events, several lines of evidence suggest that vascularcell integrin α_(v)β₃ plays a relatively late role in this process.First, immunohistological analysis reveals that expression of α_(v)β₃ onvascular cells reached a maximum 12-24 hours after the induction ofangiogenesis with bFGF. Secondly, antagonists of α_(v)β₃ perturbangiogenesis induced by multiple activators suggesting that thisreceptor is involved in common pathway downstream from perhaps allprimary signaling events leading to angiogenesis. Thirdly, mAb LM609 orcyclic peptide treated CAMs did not show a significant increase inapoptosis as measured by DNA laddering until 48 hours post treatmentwith these antagonists. Finally, antagonists of α_(v)β₃ promoteapoptosis of vascular cells that have already been induced to enter thecell cycle.

The results presented herein provide the first direct evidence thatintegrin ligation events can regulate cell survival in vivo. It istherefore hypothesized that once angiogenesis begins, individualvascular cells divide and begin to move toward the angiogenic source,after which, α_(v)β₃ ligation provides a signal allowing continued cellsurvival which leads to differentiation and the formation of matureblood vessels. However, if α_(v)β₃ ligation is prevented then the cellsfail to receive this molecular cue and the cells go into apoptosis bydefault. This hypothesis would also predict that after differentiationhas occurred mature blood vessels no longer require α_(v)β₃ signalingfor survival and thus are refractory to antagonists of this integrin.

Finally, the results presented herein provide evidence that antagonistsof integrin α_(v)β₃ may provide a powerful therapeutic approach for thetreatment of neoplasia or other diseases characterized by angiogenesis.First, antagonists of α_(v)β₃ disrupt newly forming blood vesselswithout affecting the pre-existing vasculature. Second, theseantagonists had no significant effect on chick embryo viability,suggesting they are non-toxic. Third, angiogenesis was significantlyblocked regardless of the angiogenic stimuli. Finally, systemicadministration of α_(v)β₃ antagonists causes dramatic regression ofvarious histologically distinct human tumors.

13. Preparation of Organic Molecule 3 Antagonists

The synthesis of organic α_(v)β₃ antagonist Compounds 7 (96112), 9(99799), 10 (96229), 12 (112854), 14 (96113), 15 (79959), 16 (81218), 17(87292) and 18 (87293) is described below and is also shown in the notedfigures. Organic antagonists are also referred to by the numbers inparentheses. The resultant organic molecules, referred to as organicmimetics of this invention as previously defined, are then used in themethods for inhibiting α_(v)β₃-mediated angiogenesis as described inExample 11.

For each of the syntheses described below, optical rotations weremeasured on Perkin-Elmer 241 spectrophotometer UV and visible spectrawere recorded on a Beckmann DU-70 spectrometer. ¹H and ¹³C NMR spectrawere recorded at 400 and 500 MHz on Bruker AMX-400 and AMX-500spectrometer. High-resolution mass spectra (HRMS) were recorded on a VGZAB-ZSE mass spectrometer under fast atom bombardment (FAB) conditions.Column chromatography was carried out with silica gel of 70-230 mesh.Preparative TLC was carried out on Merck Art. 5744 (0.5 mm). Meltingpoints were taken on a Thomas Hoover apparatus.

A. Compound 1: t-Boc-L-tyrosine benzyl ester as Illustrated in FIG. 38

To a solution of N-(tert-butoxycarbonyl)-L-tyrosine(t-Boc-L-tyrosine)(1.0 equivalents; Aldrich) in 0.10 M (M) methylene chloride was addeddicyclohexylcarbodiimide (DCC) (1.5 equivalents) at 25 C. and allowed tostir for 1 hour. Next, 1.5 equivalents benzyl alcohol was added and themixture was stirred for an additional 12 hours at 25 C. The reactionmixture was then diluted with ethyl acetate (0.10 M) and washed twice(2×) with water, once (1×) with brine and dried over magnesium sulfate.The solvent was then removed in vacuo and the crude product was thenpurified by silica gel column chromatography. Compound 1,t-Boc-L-tyrosine benzyl ester can also be commercially purchased fromSigma.

B. Compound 2:(S)-3-(4-(4-Bromobutyloxy)phenyl-2-N-tert-butyloxycarbonyl-propionicAcid Benzyl Ester as Illustrated in FIG. 38 Step i

A mixture of t-Boc-L-tyrosine benzyl ester (2 grams, 5.38 mmol;synthesized as described above), 1,4-dibromobutane (1.9 ml, 16.2 mmol;Aldrich), potassium carbonate (5 g) and 18-crown-6 (0.1 g; Aldrich), washeated at 80 C. for 12 hours. After cooling, the precipate was filteredoff and the reaction mixture was evaporated to dryness in vacuo. Thecrude product was then purified by crystallization using 100% hexane toyield 2.5 g (92%) of Compound 2.

C. Compound 3:(S)-3-(4-(4-Azidobutyloxy)phenyl-2-N-tert-butyloxycarbonyl-propionicAcid Benzyl Ester as Illustrated in FIG. 38 Step ii

Compound 2 (2.5 g, 4.9 mmol) was stirred with sodium azide (1.6 g, 25mmol) in dimethylformamide (DMF) (20 ml) at 25 C. for 12 hours. Thesolvent was then evaporated and the residue was treated with water(approx 10 ml) and extracted twice with ethyl acetate. The organiclayers were combined, dried via magnesium sulfate and evaporated toyield 2.0 grams (90%) of Compound 3 as a colorless syrup (FAB-MS: 469(M+H⁺).

D. Compound 4: (S)-3-(4-(4-Azidobutyloxy)phenyl-2-amino-propionic acidbenzyl ester as illustrated in FIG. 38 step iii

Compound 3 (2.0 g (4.4 mmol)) was dissolved in trifluoroacetic acid(TFA; 2 ml) and stirred for 3 hours at room temperature. Evaporation invacuo yielded 1.6 grams (quantitative) of Compound 4 as a colorlesssyrup that was used without further purification for the next step.FAB-MS: 369 (M+H⁺).

E. Compound 5:(S)-3-(4-(4-Azidobutyloxy)phenyl-2-butylsulfonamido-propionic acidbenzyl ester as illustrated in FIG. 38 step iv

A mixture of Compound 4 (1.6 g; 4.3 mmol), butane sulfonic acid chloride(0.84 ml; 6.6 mmol) and triethyl amine (1.5 equivalents) were stirred inmethylene chloride (20 ml) for 12 hours at room temperature. Thereaction mixture was then evaporated and the residue was dissolved inethylacetate, washed with dilute HCl, aqueous sodium bicarbonate andwater. After evaporation to dryness the crude product was purified byflash chromatography (silica gel, toluene/ethylacetate 15:1) to yield1.4 grams (67%) of Compound 5 as an amorphous solid.

F. Compound 6:(S)-3-(4-(4-Aminobutyloxy)phenyl-2-butylsulfonamido-propionic Acid asIllustrated in FIG. 38 Step v

Compound 5 (1.3 g (2.6 mmol) was dissolved in 20 ml of ethylacetate/methanol/water 5/3/1 and 0.2 ml trifluoroacetic acid (TFA) andhydrogenated under hydrogen (1 atmosphere; Parr Shaker apparatus) at 25C. in the presence of 100 mg palladium (10% on charcoal). After 3 hours,the catalyst was filtered off and the solvent was evaporated to yieldCompound 6 as an oily residue. After lyophilization from water 1.0 gram(quantitative) of Compound 6 was obtained as a white powder. FAB-MS: 373(M⁺H⁺).

G. Compound 7:(S)-3-(4-(4-Guanidinobutyloxy)phenyl-2-butylsulfonamido-propionic Acidas Illustrated in FIG. 38 Step vi

Compound 6 (200 mg; 0.5 mmol), 3,5-dimethylpyrazol-1-carboxamidinenitrate (DPFN) (170 mg; 0.8 mmol; Aldrich Chemical Company) andtriethylamine (0.15 ml, 1.0 mmol) in dimethylformamide (DMF; 5 ml) wereheated at 60 C. for 12 hours. After cooling, the solvent was evaporatedin vacuo, and the residue was purified by HPLC (Lichrocart RP-18,gradient acetonitrile/ water+0.3% TFA 99:1 to 1:99) to yield 50 mg (25%)of Compound 7 as a white, amorphous powder, after lyophilization.FAB-MS: 415 (M⁺H⁺), m.p.: 70 C.

H. Compound 8:(S)-3-(4-(4-Aminobutyloxy)phenyl-2-N-tert.butyloxycarbonyl-propionicAcid as Illustrated in FIG. 39 Step iii

Compound 3 (0.5 g (1.07 mmol) was dissolved in 10 ml of ethyl acetate/methanol/ water 5/3/1 and 0.1 ml trifluoroacetic acid (TFA) andhydrogenated under hydrogen (1 atmosphere; Parr Shaker apparatus) at 25C. in the presence of 30 mg palladium (10% on charcoal). After 3 hours,the catalyst was filtered off and the solvent was evaporated to yieldCompound 8 as an oily residue. After lyophilization from water 370milligram (quantitative) of Compound 8 was obtained as a white powder.FAB-MS: 353 (M⁺H⁺).

I. Compound 9:(S)-3-(4-(4-Guanidinobutyloxy)phenyl-2-N-tert.butyloxycarbonyl-propionicAcid as Illustrated in FIG. 39 Step iv

Compound 8 (200 mg; 0.5 mmol), 3,5-dimethylpyrazol-1-carboxamidinenitrate (DPFN) (170 mg; 0.8 mmol; Aldrich Chemical Company) andtriethylamine (0.15 ml, 1.0 mmol) in dimethylformamide (DMF; 5 ml) wereheated at 60 C. for 12 hours. After cooling, the solvent was evaporatedin vacuo, and the residue was purified by HPLC (Lichrocart RP-18,gradient acetonitrile/water+0.3% TFA 99:1 to 1:99) to yield 160 mg (90%)of Compound 9 as a white, amorphous powder, after lyophilization.FAB-MS: 395 (M⁺H⁺).

J. Compound 10:(R)-3-(4-(4-Guanidinobutyloxy)phenyl-2-butylsulfonamido-propionic Acidas Illustrated in FIG. 40 Steps i-vi

The identical reaction sequence to synthesize Compound 7 was used toprepare the D-tyrosine analog 10 of which 205 mg were obtained as awhite amorphous material FAB-MS: 415 (M⁺H⁺) as follows usingintermediate Compounds 100-600 to form Compound 10.

1) Compound 100: t-Boc-D-tyrosine benzyl ester as Illustrated in FIG. 40

To a solution of N-(tert-butoxycarbonyl)D-tyrosine(t-Boc-L-tyrosine)(1.0 equivalents; Aldrich) in 0.10 M methylene chloride was addeddicyclohexylcarbodiimide (DCC) (1.5 equivalents) at 25 C. and allowed tostir for 1 hour. Next, 1.5 equivalents benzyl alcohol was added and themixture was stirred for an additional 12 hours at 25 C. The reactionmixture was then diluted with ethyl acetate (0.10 M) and washed 2× withwater, 1× with brine and dried over magnesium sulfate. The solvent wasthen removed in vacuo and the crude product was then purified by silicagel column chromatography.

2) Compound 200:(R)-3-(4-(4-Bromobutyloxy)phenyl-2-N-tert-butyloxycarbonyl-propionicacid benzyl ester as illustrated in FIG. 40 Step i

A mixture of t-Boc-D-tyrosine benzyl ester (2 grams, 5.38 mmol;synthesized as described above), 1,4-dibromobutane (1.9 ml, 16.2 mmol;Aldrich), potassium carbonate (5 g) and 18-crown-6 (0.1 g; Aldrich), washeated at 80 C. for 12 hours. After cooling, the precipate was filteredoff and the reaction mixture was evaporated to dryness in vacuo. Thecrude product was then purified by crystallization using 100% hexane toyield 2.5 g (92%) of Compound 200.

3) Compound 300:(R)-3-(4-(4-Azidobutyloxy)phenyl-2-N-tert-butyloxycarbonyl-propionicacid benzyl ester as illustrated in FIG. 40 Step ii

Compound 200 (2.5 g, 4.9 mmol) was stirred with sodium azide (1.6 g, 25mmol) in dimethylformamide (DMF) (20 ml) at 25 C. for 12 hours. Thesolvent was then evaporated and the residue was treated with water(approx 10 ml) and extracted twice with ethyl acetate. The organiclayers were combined, dried via magnesium sulfate and evaporated toyield 2.0 grams (90%) of Compound 300 as a colorless syrup (FAB-MS: 469(M⁺H⁺).

4) Compound 400: (R)-3-(4-(4-Azidobutyloxy)phenyl-2-amino-propionic AcidBenzyl Ester as Illustrated in FIG. 40 Step iii

Compound 300 (2.0 g (4.4 mmol)) was dissolved in trifluoroacetic acid(TFA; 2 ml) and stirred for 3 hours at room temperature. Evaporation invacuo yielded 1.6 grams (quantitative) of Compound 400 as a colorlesssyrup that was used without further purification for the next step.FAB-MS: 369 (M⁺H⁺).

5) Compound 500:(R)-3-(4-(4-Azidobutyloxy)phenyl-2-butylsulfonamido-propionic AcidBenzyl Ester as Illustrated in FIG. 40 Step iv

A mixture of Compound 400 (1.6 g; 4.3 mmol), butane sulfonic acidchloride (0.84 ml; 6.6 mmol) and triethyl amine (1.5 equivalents) werestirred in methylene chloride (20 ml) for 12 hours at room temperature.The reaction mixture was then evaporated and the residue was dissolvedin ethylacetate, washed with dilute HCl, aqueous sodium bicarbonate andwater. After evaporation to dryness the crude product was purified byflash chromatography (silica gel, toluene/ethylacetate 15:1) to yield1.4 grams (67%) of Compound 500 as an amorphous solid.

6) Compound 600:(R)-3-(4-(4-Aminobutyloxy)phenyl-2-butylsulfonamido-propionic Acid asIllustrated in FIG. 40 Step v

Compound 500 (1.3 g (2.6 mmol) was dissolved in 20 ml of ethylacetate/methanol/water 5/3/1 and 0.2 ml trifluoroacetic acid (TFA) andhydrogenated under hydrogen (1 atmosphere; Parr Shaker apparatus) at 25C. in the presence of 100 mg palladium (10% on charcoal). After 3 hours,the catalyst was filtered off and the solvent was evaporated to yieldCompound 600 as an oily residue. After lyophilization from water 1.0gram (quantitative) of Compound 600 was obtained as a white powder.FAB-MS: 373 (M⁺H⁺).

7) Compound 10:(R)-3-(4-(4-Guanidinobutyloxy)phenyl-2-butylsulfonamido-propionic Acidas Illustrated in FIG. 40 Step vi

Compound 600 (200 mg; 0.5 mmol), 3,5-dimethylpyrazol-1-carboxamidinenitrate (DPFN) (170 mg; 0.8 mmol; Aldrich Chemical Company) andtriethylamine (0.15 ml, 1.0 mmol) in dimethylformamide (DMF; 5 ml) wereheated at 60 C. for 12 hours. After cooling, the solvent was evaporatedin vacuo, and the residue was purified by HPLC (Lichrocart RP-18,gradient acetonitrile/water+0.3% TFA 99:1 to 1:99) to yield 50 mg (25%)of Compound 10 as a white, amorphous powder, after lyophilization.FAB-MS: 415 (M⁺H⁺), m.p.: 70 C.

K. Compound 11:(S)-3-(4-(4-Azidobutyloxy)phenyl-2-(10-camphorsulfonamido)-propionicAcid Benzyl Ester as Illustrated in FIG. 4

A mixture of compound 4 (1.0 g; 2.7 mmol), 10-camphorsulfonic acidchloride (6.6 mmol; Aldrich Chemical Company) and triethyl amine (1.5equivalents) were stirred in methylene chloride (20 mL) for 12 hours atroom temperature. The reaction mixture was then evaporated and theresidue was dissolved in ethylacetate, washed with dilute HCl, aqueoussodium bicarbonate and water. After evaporation to dryness the crudeproduct was purified by flash chromatography (silica gel,toluene/ethylacetate 15:1) to yield 1.4 grams (67%) of compound 11 as anamorphous solid.

L. Compound 12:(S)-3-(4-(4-Guanidinobutyloxy)phenyl-2-(10-camphorsulfonamido)-propionicAcid as Illustrated in FIG. 41 steps i-ii

Compound 12 was obtained after hydrogenation and guanylation of Compound11 according to the following conditions:

Step i: Compound 11 (1.3 g (2.6 mmol) was dissolved in 20 ml of ethylacetate/methanol/water 5/3/1 and 0.2 ml trifluoroacetic acid (TFA) andhydrogenated under hydrogen (1 atmosphere; Parr Shaker apparatus) at 25C. in the presence of 100 mg palladium (10% on charcoal). After 3 hours,the catalyst was filtered off and the solvent was evaporated to yieldthe intermediate amine as an oily residue. After lyophilization fromwater 1.0 gram (quantitative) of the intermediate amine was obtained asa white powder, which was carried on as follows:

Step ii: The above formed intermediate amine compound (200 mg; 0.5mmol), 3,5-dimethylpyrazol-1-carboxamidine nitrate (DPFN) (170 mg; 0.8mmol; Aldrich Chemical Company) and triethylamine (0.15 ml, 1.0 mmol) indimethylformamide (DMF; 5 ml) were heated at 60 C. for 12 hours. Aftercooling, the solvent was evaporated in vacuo, and the residue waspurified by HPLC (Lichrocart RP-18, gradient acetonitrile/water+0.3% TFA99:1 to 1:99) to yield 50 mg (25%) of Compound 12 as a white, amorphouspowder, after lyophilization. FAB-MS: 509.6 (M⁺H⁺).

M. Compound 13:(S)-3-(4-(5-Bromopentyloxy)phenyl-2-N-tert.butyloxycarbonyl-propionicAcid Benzyl Ester as Illustrated in FIG. 41

A mixture of t-Boc-L-tyrosine benzyl ester (4.5 grams, 12.1 mmol;Compound 1 synthesized as described above), 1,5-dibromopentane (5 ml,36.7 mmol; Aldrich), potassium carbonate (10 g) and 18-crown-6 (0.25 g;Aldrich), was heated at 80 C. for 12 hours. After cooling, the precipatewas filtered off and the reaction mixture was evaporated to dryness invacuo. The crude product was then purified by crystallization using 100%hexane to yield 5.35 g (85%) of Compound 13.

N. Compound 14:(S)-3-(4-(5-Guanidinopentyloxy)phenyl-2-butylsulfonamido-propionic Acidas Illustrated in FIG. 41 steps i-v

The 5 step reaction sequence of bromine-azide-exchange, Boc-cleavage,sulfonylation with butane sulfonic acid chloride, hydrogenation andguanylation with DPFN was carried out identically to the aboveprocedures using intermediate Compounds 1-6 to form Compound 7 or theprocedures using Compounds 100-600 to form Compound 10, as disclosedabove. Compound 14 was obtained as a white powder FAB-MS: 429 (M⁺H⁺).

O. Compound 15:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-amino-ethyl)phenoxy)methyl-2-oxazolidinone,dihydrochloride as Shown in FIG. 42

1) Synthesis of Starting Material2-N-BOC-amino-3-(4-hydroxy-phenyl)propionate for Compound 15

The starting material 2-N-BOC-amino-3-(4-hydroxy-phenyl)propionate wasobtained via esterification of (D or L),N-(tert-butoxycarbonyl)-L(D)-tyrosine (t-Boc-L(D)-tyrosine) (1.0equivalents; Sigma) in 0.10 M methanol and dilute 1% HCl. The reactionmixture was stirred at 25 C. for 12 hours and then neutralized viapotassium carbonate and then diluted with ethyl acetate (0.10 M) andwashed 2× with water, 1× with brine and dried over magnesium sulfate.The solvent was then removed in vacuo and the crude product was thenpurified by silica gel column chromatography to obtain2-N-BOC-amino-3-(4-hydroxy-phenyl)propionate.

2) Synthesis of starting material3-D-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone forCompound 15: 3-step procedure as follows:

p-amino-benzonitrile (1.0 equivalents; Aldrich) in methylene chloride(0.10 M) was stirred with 2,3-epoxypropanol (1.0 equivalents; Aldrich)for 12 hours at 25 C. The solvent was next removed in vacuo and thecrude 4-(2,3-dihydroxypropylamino)benzonitrile was carried onto the nextstep as follows:

4-(2,3-dihydroxypropylamino)benzonitrile (1.0 equivalents; as describedabove), in dimethylformamide (0.10 M), at 25 C., was stirred withdiethyl carbonate (1.1 equivalents; Aldrich) and potassium tert-butylate(1.1 equivalents; Aldrich) at 110 C. for 6 hours. Next, the reactionmixture was diluted with ethyl acetate (0.10 M) and washed 2× withwater, 1× with brine and dried over magnesium sulfate. The solvent wasthen removed in vacuo and the crude product was then purified by silicagel column chromatography to obtain3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine and carried onto thenext step as follows:

3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine (1.0 equivalents; asdescribed above), in methylene chloride (0.10 M) at 25 C. was stirredwith 1.1 equivalents hydrogen sulfide, 1.1 equivalents methyl iodide,and 1.1 equivalents ammonium acetate. The reaction mixture was stirredfor 6 hours and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain the amidine which was carriedonto the next step as follows:

1.0 equivalents of the amidine, synthesized as described above, wasprotected with 1.1 equivalents of BOC-ON(2-(BOC-oxyimino)-2-phenylacetonitrile; Aldrich) in methylene chloride(0.10 M) at 25 C. and stirred for 6 hours. Next, the reaction mixturewas diluted with ethyl acetate (0.10 M) and washed 2× with water, 1×with brine and dried over magnesium sulfate. The solvent was thenremoved in vacuo and the crude product was then esterified in 0.10 Mmethylene chloride and 1.1 equivalents methanesulfonyl chloride. Thereaction mixture was stirred at 0 C. for 6 hours and then quenched withwater (5 equivalents) and then diluted with ethyl acetate (0.10 M) andwashed 2× with water, 1× with brine and dried over magnesium sulfate.The solvent was then removed in vacuo and the crude product was thenpurified by silica gel column chromatography to obtain3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone.

3) Coupling of intermediates2-N-BOC-amino-3-(4-hydroxy-phenyl)propionate with3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone toform protected form of Compound 15,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2N-BOC-aminoethyl)phenyoxylmethyl-2-oxazolidinone

A mixture of 1.9 grams 2-N-BOC-amino-3-(4-hydroxy-phenyl)propionate (asdescribed above), 20 ml dimethylformamide (DMF) and NaH (1.0equivalent), was stirred for 30 minutes at room temperature. Afterstirring, 1.8 grams3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone (asdescribed above) in 10 ml dimethylformamide (DMF) was added and stirredagain for 15 minutes at room temperature. The reaction mixture was thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then purified by silica gel columnchromatography to obtain protected form of Compound 15,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2N-BOC-aminoethyl)phenyoxylmethyl-2-oxazolidinonewhich was carried onto the next step.

4) Deprotection of protected form of compound 15 to form Compound 15:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-amino-ethyl)phenoxy)methyl-2-oxazolidinone,dihydrochloride, FIG. 42

Treatment of the protected form of Compound 15,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2N-BOC-aminoethyl)phenyoxylmethyl-2-oxazolidinone(1.0 equivalents; synthesized as described above), with 4 ml 2N NaOH for4 hours at room temperature. The mixture was then followed with 40 ml 2NHCl-solution in dioxane added dropwise at 0 C. to 25 C. for 3 hours. Thereaction mixture was then quenched with sodium bicarbonate (5equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain Compound 15:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-amino-ethyl)phenoxy)methyl-2-oxazolidinone,dihydrochloride; m.p. 165C(d).

P. Compound 16:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-N-butylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinoneas Shown in FIG. X (Old 14)

1) Synthesis of starting material2-N-butylsulfonylamino-3-(4-hydroxy-phenyl)propionate for Compound 16

The starting material2-N-butylsulfonylamino-3-(4-hydroxy-phenyl)propionate was obtained viaesterification of ((D or L) tyrosine) (1.0 equivalents; Sigma) in 0.10 Mmethanol and dilute 1% HCl. The reaction mixture was stirred at 25 C.for 12 hours and then neutralized via potassium carbonate and thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then carried on as follows:

A mixture of the above compound (4.3 mmol), butane sulfonic acidchloride (6.6 mmol) and triethyl amine (1.5 equivalents) were stirred inmethylene chloride (20 ml) for 12 hours at room temperature. Thereaction mixture was then evaporated and the residue was dissolved inethylacetate, washed with dilute HCl, aqueous sodium bicarbonate andwater. After evaporation to dryness the crude product was purified byflash chromatography (silica gel, toluene/ethylacetate 15:1) to yieldthe title compound.

2) Synthesis of starting material3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone forCompound 16: 3-step procedure as follows:

p-amino-benzonitrile (1.0 equivalents; Aldrich) in methylene chloride(0.10 M) was stirred with 2,3-epoxypropanol (1.0 equivalents; Aldrich)for 12 hours at 25 C. The solvent was next removed in vacuo and thecrude 4-(2,3-dihydroxypropylamino)benzonitrile was carried onto the nextstep as follows:

4-(2,3-dihydroxypropylamino)benzonitrile (1.0 equivalents; as describedabove), in dimethylformamide (0.10 M), at 25 C., was stirred withdiethyl carbonate (1.1 equivalents; Aldrich) and potassium tert-butylate(1.1 equivalents; Aldrich) at 110 C. for 6 hours. Next, the reactionmixture was diluted with ethyl acetate (0.10 M) and washed 2× withwater, 1× with brine and dried over magnesium sulfate. The solvent wasthen removed in vacuo and the crude product was then purified by silicagel column chromatography to obtain3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine and carried onto thenext step as follows:

3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine (1.0 equivalents; asdescribed above), in methylene chloride (0.10 M) at 25 C. was stirredwith 1.1 equivalents hydrogen sulfide, 1.1 equivalents methyl iodide,and 1.1 equivalents ammonium acetate. The reaction mixture was stirredfor 6 hours and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain the amidine which was carriedonto the next step as follows:

1.0 equivalents of the amidine, synthesized as described above, wasprotected with 1.1 equivalents of BOC-ON(2-(BOC-oxyimino)-2-phenylacetonitrile; Aldrich) in methylene chloride(0.10 M) at 25 C. and stirred for 6 hours. Next, the reaction mixturewas diluted with ethyl acetate (0.10 M) and washed 2× with water, 1×with brine and dried over magnesium sulfate. The solvent was thenremoved in vacuo and the crude product was then esterified in 0.10 Mmethylene chloride and 1.1 equivalents methanesulfonyl chloride. Thereaction mixture was stirred at 0 C. for 6 hours and then quenched withwater (5 equivalents) and then diluted with ethyl acetate (0.10 M) andwashed 2× with water, 1× with brine and dried over magnesium sulfate.The solvent was then removed in vacuo and the crude product was thenpurified by silica gel column chromatography to obtain3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone.

3) Coupling of intermediates2-N-butylsulfonylamino-3-(4-hydroxy-phenyl)propionate with3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone toform Protected form of Compound 16,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-butylsulfonylaminoethyl)rphenyoxylmethyl-2-oxazolidinone

A mixture of 1.9 grams2-N-butylsulfonylamino-3-(4-hydroxy-phenyl)propionate (as describedabove), 20 ml dimethylformamide (DMF) and NaH (1.0 equivalent), wasstirred for 30 minutes at room temperature. After stirring, 1.8 grams3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone (asdescribed above) in 10 ml dimethylformamide (DMF) was added and stirredagain for 15 minutes at room temperature. The reaction mixture was thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then purified by silica gel columnchromatography to obtain protected form of Compound 16,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-butylsulfonylaminoethyl)-phenyoxylmethyl-2-oxazolidinonewhich was carried onto the next step.

4) Deprotection of protected form of Compound 16 to Form Compound 16:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-N-butylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinone.FIG. 42

Treatment of the protected form of Compound 16, 3-(4-BOC-amidinophenyl)-5(4-(2-methoxy-carbonyl-2-N-butylsulfonylaminoethyl)phenyoxylmethyl-2-oxazolidinone(1.0 equivalents; synthesized as described above), with 4 ml 2N NaOH for4 hours at room temperature. The mixture was then followed with 40 ml 2NHCl-solution in dioxane added dropwise at 0 C. to 25 C. for 3 hours. Thereaction mixture was then quenched with sodium bicarbonate (5equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain Compound 16:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-N-butylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinone;m.p. 236-237 C.

Q. Compound 17:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-N-propyl-sulfonylaminoethyl)phenoxy)methyl-2-oxazolidinoneas shown in FIG. 42

1) Synthesis of starting material2-N-propyl-sulfonylamino-3-(4-hydroxy-phenyl)propionate for Compound 17;

The starting material2-N-propyl-sulfonylamino-3-(4-hydroxy-phenyl)propionate was obtained viaesterification of ((D or L) tyrosine) (1.0 equivalents; Sigma) in 0.10 Mmethanol and dilute 1% HCl. The reaction mixture was stirred at 25 C.for 12 hours and then neutralized via potassium carbonate and thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× brineand dried over magnesium sulfate. The solvent was then removed in vacuoand the crude product was then carried on as follows:

A mixture of the above compound (4.3 mmol), propyl sulfonic acidchloride (6.6 mmol; Aldrich) and triethyl amine (1.5 equivalents) werestirred in methylene chloride (20 ml) for 12 hours at room temperature.The reaction mixture was then evaporated and the residue was dissolvedin ethylacetate, washed with dilute HCl, aqueous sodium bicarbonate andwater. After evaporation to dryness the crude product was purified byflash chromatography (silica gel, toluene/ethylacetate 15:1) to yieldthe title compound.

2) Synthesis of starting material3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone forCompound 17: 3-step Procedure as Follows:

p-amino-benzonitrile (1.0 equivalents; Aldrich) in methylene chloride(0.10 M) was stirred with 2,3-epoxypropanol (1.0 equivalents; Aldrich)for 12 hours at 25 C. The solvent was next removed in vacuo and thecrude 4-(2,3-dihydroxypropylamino)benzonitrile was carried onto the nextstep as follows:

4-(2,3-dihydroxypropylamino)benzonitrile (1.0 equivalents; as describedabove), in dimethylformamide (0.10 M), at 25 C., was stirred withdiethyl carbonate (1.1 equivalents; Aldrich) and potassium tert-butylate(1.1 equivalents; Aldrich) at 110 C. for 6 hours. Next, the reactionmixture was diluted with ethyl acetate (0.10 M) and washed 2× withwater, 1× with brine and dried over magnesium sulfate. The solvent wasthen removed in vacuo and the crude product was then purified by silicagel column chromatography to obtain3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine and carried onto thenext step as follows:

3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine (1.0 equivalents; asdescribed above), in methylene chloride (0.10 M) at 25 C. was stirredwith 1.1 equivalents hydrogen sulfide, 1.1 equivalents methyl iodide,and 1.1 equivalents ammonium acetate. The reaction mixture was stirredfor 6 hours and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain the amidine which was carriedonto the next step as follows:

1.0 equivalents of the amidine, synthesized as described above, wasprotected with 1.1 equivalents of BOC-ON(2-(BOC-oxyimino)-2-phenylacetonitrile; Aldrich) in methylene chloride(0.10 M) at 25 C. and stirred for 6 hours. Next, the reaction mixturewas diluted with ethyl acetate (0.10 M) and washed 2× with water, 1×with brine and dried over magnesium sulfate. The solvent was thenremoved in vacuo and the crude product was then esterified in 0.10 Mmethylene chloride and 1.1 equivalents methanesulfonyl chloride. Thereaction mixture was stirred at 0 C. for 6 hours and then quenched withwater (5 equivalents) and then diluted with ethyl acetate (0.10 M) andwashed 2× with water, 1× with brine and dried over magnesium sulfate.The solvent was then removed in vacuo and the crude product was thenpurified by silica gel column chromatography to obtain3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone.

3) Coupling of intermediates2-N-propyl-sulfonylamino-3-(4-hydroxy-phenyl)propionate with3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone toform protected form of Compound 17,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-propyl-sulfonylaminoethyl)-phenyoxylmethyl-2-oxazolidinone

A mixture of 1.9 grams2-N-propyl-sulfonylamino-3-(4-hydroxy-phenyl)propionate (as describedabove), 20 ml dimethylformamide (DMF) and NaH (1.0 equivalent), wasstirred for 30 minutes at room temperature. After stirring, 1.8 grams3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone (asdescribed above) in 10 ml dimethylformamide (DMF) was added and stirredagain for 15 minutes at room temperature. The reaction mixture was thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then purified by silica gel columnchromatography to obtain protected form of Compound 17,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-propyl-sulfonylaminoethyl)-phenyoxylmethyl-2-oxazolidinonewhich was carried onto the next step.

4) Deprotection of protected form of compound 17 to Form Compound 17:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-N-propylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinone.FIG. 42

Treatment of the protected form of Compound 17,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-propylsulfonylaminoethyl)phenyoxylmethyl-2-oxazolidinone(1.0 equivalents; synthesized as described above), with 4 ml 2N NaOH for4 hours at room temperature. The mixture was then followed with 40 ml 2NHCl-solution in dioxane added dropwise at 0 C. to 25 C. for 3 hours. Thereaction mixture was then quenched with sodium bicarbonate (5equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain Compound 17:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-N-propylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinone;m.p. 200 C. (d).

R. Compound 18:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-N-ethyl-sulfonylaminoethyl)phenoxy)methyl-2-oxazolidinoneas Shown in FIG. 42

1) Synthesis of starting material2-N-ethyl-sulfonylamino-3-(4-hydroxy-phenyl)propionate for Compound 18

The starting material2-N-ethyl-sulfonylamino-3-(4-hydroxy-phenyl)propionate was obtained viaesterification of ((D or L) tyrosine) (1.0 equivalents; Sigma) in 0.10 Mmethanol and dilute 1% HCl. The reaction mixture was stirred at 25 C.for 12 hours and then neutralized via potassium carbonate and thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then carried on as follows:

A mixture of the above compound (4.3 mmol), ethyl sulfonic acid chloride(6.6 mmol; Aldrich) and triethyl amine (1.5 equivalents) were stirred inmethylene chloride (20 ml) for 12 hours at room temperature. Thereaction mixture was then evaporated and the residue was dissolved inethylacetate, washed with dilute HCl, aqueous sodium bicarbonate andwater. After evaporation to dryness the crude product was purified byflash chromatography (silica gel, toluene/ethylacetate 15:1) to yieldthe title compound.

2) Synthesis of starting material3-D-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone forCompound 18: 3-step Procedure as Follows:

p-amino-benzonitrile (1.0 equivalents; Aldrich) in methylene chloride(0.10 M) was stirred with 2,3-epoxypropanol (1.0 equivalents; Aldrich)for 12 hours at 25 C. The solvent was next removed in vacuo and thecrude 4-(2,3-dihydroxypropylamino)benzonitrile was carried onto the nextstep as follows:

4-(2,3-dihydroxypropylamino)benzonitrile (1.0 equivalents; as describedabove), in dimethylformamide (0.10 M), at 25 C., was stirred withdiethyl carbonate (1.1 equivalents; Aldrich) and potassium tert-butylate(1.1 equivalents; Aldrich) at 110 C. for 6 hours. Next, the reactionmixture was diluted with ethyl acetate (0.10 M) and washed 2× withwater, 1× with brine and dried over magnesium sulfate. The solvent wasthen removed in vacuo and the crude product was then purified by silicagel column chromatography to obtain3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine and carried onto thenext step as follows:

3-(4-cyanophenyl)-5-hydroxymethyl-2-oxazolidine (1.0 equivalents; asdescribed above), in methylene chloride (0.10 M) at 25 C. was stirredwith 1.1 equivalents hydrogen sulfide, 1.1 equivalents methyl iodide,and 1.1 equivalents ammonium acetate. The reaction mixture was stirredfor 6 hours and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain the amidine which was carriedonto the next step as follows:

1.0 equivalents of the amidine, synthesized as described above, wasprotected with 1.1 equivalents of BOC-ON(2-(BOC-oxyimino)-2-phenylacetonitrile; Aldrich) in methylene chloride(0.10 M) at 25 C. and stirred for 6 hours. Next, the reaction mixturewas diluted with ethyl acetate (0.10 M) and washed 2× with water, 1×with brine and dried over magnesium sulfate. The solvent was thenremoved in vacuo and the crude product was then esterified in 0.10 Mmethylene chloride and 1.1 equivalents methanesulfonyl chloride. Thereaction mixture was stirred at 0 C. for 6 hours and then quenched withwater (5 equivalents) and then diluted with ethyl acetate (0.10 M) andwashed 2× with water, 1× with brine and dried over magnesium sulfate.The solvent was then removed in vacuo and the crude product was thenpurified by silica gel column chromatography to obtain3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone.

3) Coupling of intermediates2-N-ethyl-sulfonylamino-3-(4-hydroxy-phenyl)propionate with3-n-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone toform protected form of Compound 18,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-ethyl-sulfonylaminoethyl)-phenyoxylmethyl-2-oxazolidinone

A mixture of 1.9 grams2-N-ethyl-sulfonylamino-3-(4-hydroxy-phenyl)propionate (as describedabove), 20 ml dimethylformamide (DMF) and NaH (1.0 equivalent), wasstirred for 30 minutes at room temperature. After stirring, 1.8 grams3-p-N-BOC-amidino-phenyl-5-methanesulfonyloxy-methyl-2-oxazolidinone (asdescribed above) in 10 ml dimethylformamide (DMF) was added and stirredagain for 15 minutes at room temperature. The reaction mixture was thendiluted with ethyl acetate (0.10 M) and washed 2× with water, 1× withbrine and dried over magnesium sulfate. The solvent was then removed invacuo and the crude product was then purified by silica gel columnchromatography to obtain protected form of Compound 18,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-ethyl-sulfonylaminoethyl)-phenyoxylmethyl-2-oxazolidinonewhich was carried onto the next step.

4) Deprotection of protected form of Compound 18 to form Compound 18:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-N-ethylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinone.FIG. 42

Treatment of the protected form of Compound 18,3-(4-BOC-amidinophenyl)-5-(4-(2-methoxy-carbonyl-2-N-ethylsulfonylaminoethyl)phenyoxylmethyl-2-oxazolidinone(1.0 equivalents; synthesized as described above), with 4 ml 2N NaOH for4 hours at room temperature. The mixture was then followed with 40 ml 2NHCl-solution in dioxane added dropwise at 0 C. to 25 C. for 3 hours. Thereaction mixture was then quenched with sodium bicarbonate (5equivalents) and then diluted with ethyl acetate (0.10 M) and washed 2×with water, 1× with brine and dried over magnesium sulfate. The solventwas then removed in vacuo and the crude product was then purified bysilica gel column chromatography to obtain Compound 18:3-(4-amidinophenyl)-5-(4-(2-carboxy-2-N-ethylsulfonylaminoethyl)phenoxy)methyl-2-oxazolidinone;m.p. 212 C. (d).

14. Inhibition of Growth Factor-Induced Anaiogenesis as Measured in theCAM Assay with by Intravenous Application of α_(v)β₃ Ligand OrganicMimetics

The effect on growth factor-induced angiogenesis with organic mimeticsof an α_(v)β₃ ligand intravenously injected into the CAM preparation wasalso evaluated for use in this invention.

The 10 day old CAM preparation was used as previously described inExample 5A. Twenty-four hours after bFGF-induced angiogenesis wasinitiated, the organic mimetics referred to as compounds 16 (81218), 17(87292) and 18 (87293) were separately intravenously injected into theCAM preparation in a volume of 100 ul at a concentration of 1 mg/ml (100ug/embryo) in 20% tetraglycol-PBS at pH 7.0. In parallel assays,compounds 7 (96112), 9 (99799), 10 (96229), 12 (112854) and 14 (96113)were similarly evaluated. The effects of the organic mimetics wereanalyzed 48 hours later where quantification was performed by countingthe number of blood vessel branch points in the area of the filter discin a double blind approach.

The results are respectively shown in FIGS. 43 and 44. In FIG. 43,compounds 14 (96113), 10 (96229), 9 (99799) and 12 (112854), indecreasing order of inhibition, were effective at reducing the number ofbranch points of new blood vessels compared to control bFGF inductionand compared to compound 7 (96112). In FIG. 44, compounds 17 (87292) and18 (87293) exhibited anti-angiogenic properties as compared to untreatedbFGF control and treatment with compound 16 (81218).

In a third assay, organic compounds 7 (96112), 10 (96229) and 14 (96113)were assessed as inhibitors of bFGF-induced angiogenesis along withpeptides 69601 and 66203. For this assay, 250 ug/ml of organic compoundswere administered 18 hours after bFGF treatment as described in Example7B. The results are shown in FIG. 28 where as above, compounds 14(96113) and 10 (96229) almost completely inhibited the formation of newblood vessels induced by bFGF.

Thus, the aforementioned Examples demonstrate that integrin α_(v)β₃plays a key role in angiogenesis induced by a variety of stimuli and assuch α_(v)β₃ is a valuable therapeutic target with the α_(v)β₃antagonists of this invention for diseases characterized byneovascularization.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the cell line deposited,since the deposited embodiment is intended as a single illustration ofone aspect of the invention and any cell line that is functionallyequivalent is within the scope of this invention. The deposit ofmaterial does not constitute an admission that the written descriptionherein contained is inadequate to enable the practice of any aspect ofthe invention, including the best mode thereof, nor is it to beconstrued as limiting the scope of the claims to the specificillustration that it represents. Indeed, various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims.

45 1 6 PRT Artificial Sequence Description of Artificial Sequencepeptide 1 Gly Arg Gly Asp Phe Val 1 5 2 6 PRT Artificial SequenceDescription of Artificial Sequence peptide 2 Gly Arg Gly Asp Phe Val 1 53 6 PRT Artificial Sequence Description of Artificial Sequence peptide 3Gly Arg Gly Asp Phe Val 1 5 4 6 PRT Artificial Sequence Description ofArtificial Sequence peptide 4 Gly Arg Gly Asp Phe Val 1 5 5 5 PRTArtificial Sequence Description of Artificial Sequence peptide 5 Arg GlyAsp Phe Val 1 5 6 5 PRT Artificial Sequence Description of ArtificialSequence peptide 6 Arg Ala Asp Phe Val 1 5 7 5 PRT Artificial SequenceDescription of Artificial Sequence peptide 7 Arg Gly Asp Phe Val 1 5 815 PRT Artificial Sequence Description of Artificial Sequence peptide 8Tyr Thr Ala Glu Cys Lys Pro Gln Val Thr Arg Gly Asp Val Phe 1 5 10 15 95 PRT Artificial Sequence Description of Artificial Sequence peptide 9Arg Ala Asp Phe Val 1 5 10 6 PRT Artificial Sequence Description ofArtificial Sequence peptide 10 Ala Arg Gly Asp Phe Leu 1 5 11 6 PRTArtificial Sequence Description of Artificial Sequence peptide 11 GlyArg Gly Asp Phe Leu 1 5 12 12 PRT Artificial Sequence Description ofArtificial Sequence peptide 12 Thr Arg Gln Val Val Cys Asp Leu Gly AsnPro Met 1 5 10 13 13 PRT Artificial Sequence Description of ArtificialSequence peptide 13 Gly Val Val Arg Asn Asn Glu Ala Leu Ala Arg Leu Ser1 5 10 14 11 PRT Artificial Sequence Description of Artificial Sequencepeptide 14 Thr Asp Val Asn Gly Asp Gly Arg His Asp Leu 1 5 10 15 5 PRTArtificial Sequence Description of Artificial Sequence peptide 15 ArgGly Asp Phe Val 1 5 16 5 PRT Artificial Sequence Description ofArtificial Sequence peptide 16 Arg Gly Glu Phe Val 1 5 17 222 PRT Homosapiens 17 Lys Gly Ile Gln Glu Leu Tyr Gly Ala Ser Pro Asp Ile Asp LeuGly 1 5 10 15 Thr Gly Pro Thr Pro Thr Leu Gly Pro Val Thr Pro Glu IleCys Lys 20 25 30 Gln Asp Ile Val Phe Asp Gly Ile Ala Gln Ile Arg Gly GluIle Phe 35 40 45 Phe Phe Lys Asp Arg Phe Ile Trp Arg Thr Val Thr Pro ArgAsp Lys 50 55 60 Pro Met Gly Pro Leu Leu Val Ala Thr Phe Trp Pro Glu LeuPro Glu 65 70 75 80 Lys Ile Asp Ala Val Tyr Glu Ala Pro Gln Glu Glu LysAla Val Phe 85 90 95 Phe Ala Gly Asn Glu Tyr Trp Ile Tyr Ser Ala Ser ThrLeu Glu Arg 100 105 110 Gly Tyr Pro Lys Pro Leu Thr Ser Leu Gly Leu ProPro Asp Val Gln 115 120 125 Arg Val Asp Ala Ala Phe Asn Trp Ser Lys AsnLys Lys Thr Tyr Ile 130 135 140 Phe Ala Gly Asp Lys Phe Trp Arg Tyr AsnGlu Val Lys Lys Lys Met 145 150 155 160 Asp Pro Gly Phe Pro Lys Leu IleAla Asp Ala Trp Asn Ala Ile Pro 165 170 175 Asp Asn Leu Asp Ala Val ValAsp Leu Gln Gly Gly Gly His Ser Tyr 180 185 190 Phe Phe Lys Gly Ala TyrTyr Leu Lys Leu Glu Asn Gln Ser Leu Lys 195 200 205 Ser Val Lys Phe GlySer Ile Lys Ser Asp Trp Leu Gly Cys 210 215 220 18 193 PRT Homo sapiens18 Ile Cys Lys Gln Asp Ile Val Phe Asp Gly Ile Ala Gln Ile Arg Gly 1 510 15 Glu Ile Phe Phe Phe Lys Asp Arg Phe Ile Trp Arg Thr Val Thr Pro 2025 30 Arg Asp Lys Pro Met Gly Pro Leu Leu Val Ala Thr Phe Trp Pro Glu 3540 45 Leu Pro Glu Lys Ile Asp Ala Val Tyr Glu Ala Pro Gln Glu Glu Lys 5055 60 Ala Val Phe Phe Ala Gly Asn Glu Tyr Trp Ile Tyr Ser Ala Ser Thr 6570 75 80 Leu Glu Arg Gly Tyr Pro Lys Pro Leu Thr Ser Leu Gly Leu Pro Pro85 90 95 Asp Val Gln Arg Val Asp Ala Ala Phe Asn Trp Ser Lys Asn Lys Lys100 105 110 Thr Tyr Ile Phe Ala Gly Asp Lys Phe Trp Arg Tyr Asn Glu ValLys 115 120 125 Lys Lys Met Asp Pro Gly Phe Pro Lys Leu Ile Ala Asp AlaTrp Asn 130 135 140 Ala Ile Pro Asp Asn Leu Asp Ala Val Val Asp Leu GlnGly Gly Gly 145 150 155 160 His Ser Tyr Phe Phe Lys Gly Ala Tyr Tyr LeuLys Leu Glu Asn Gln 165 170 175 Ser Leu Lys Ser Val Lys Phe Gly Ser IleLys Ser Asp Trp Leu Gly 180 185 190 Cys 19 74 PRT Homo sapiens 19 IleCys Lys Gln Asp Ile Val Phe Asp Gly Ile Ala Gln Ile Arg Gly 1 5 10 15Glu Ile Phe Phe Phe Lys Asp Arg Phe Ile Trp Arg Thr Val Thr Pro 20 25 30Arg Asp Lys Pro Met Gly Pro Leu Leu Val Ala Thr Phe Trp Pro Glu 35 40 45Leu Pro Glu Lys Ile Asp Ala Val Tyr Glu Ala Pro Gln Glu Glu Lys 50 55 60Ala Val Phe Phe Ala Gly Asn Glu Tyr Trp 65 70 20 108 PRT Homo sapiens 20Ile Cys Lys Gln Asp Ile Val Phe Asp Gly Ile Ala Gln Ile Arg Gly 1 5 1015 Glu Ile Phe Phe Phe Lys Asp Arg Phe Ile Trp Arg Thr Val Thr Pro 20 2530 Arg Asp Lys Pro Met Gly Pro Leu Leu Val Ala Thr Phe Trp Pro Glu 35 4045 Leu Pro Glu Lys Ile Asp Ala Val Tyr Glu Ala Pro Gln Glu Glu Lys 50 5560 Ala Val Phe Phe Ala Gly Asn Glu Tyr Trp Ile Tyr Ser Ala Ser Thr 65 7075 80 Leu Glu Arg Gly Tyr Pro Lys Pro Leu Thr Ser Leu Gly Leu Pro Pro 8590 95 Asp Val Gln Arg Val Asp Ala Ala Phe Asn Trp Ser 100 105 21 122 PRTHomo sapiens 21 Glu Tyr Trp Ile Tyr Ser Ala Ser Thr Leu Glu Arg Gly TyrPro Lys 1 5 10 15 Pro Leu Thr Ser Leu Gly Leu Pro Pro Asp Val Gln ArgVal Asp Ala 20 25 30 Ala Phe Asn Trp Ser Lys Asn Lys Lys Thr Tyr Ile PheAla Gly Asp 35 40 45 Lys Phe Trp Arg Tyr Asn Glu Val Lys Lys Lys Met AspPro Gly Phe 50 55 60 Pro Lys Leu Ile Ala Asp Ala Trp Asn Ala Ile Pro AspAsn Leu Asp 65 70 75 80 Ala Val Val Asp Leu Gln Gly Gly Gly His Ser TyrPhe Phe Lys Gly 85 90 95 Ala Tyr Tyr Leu Lys Leu Glu Asn Gln Ser Leu LysSer Val Lys Phe 100 105 110 Gly Ser Ile Lys Ser Asp Trp Leu Gly Cys 115120 22 89 PRT Homo sapiens 22 Phe Asn Trp Ser Lys Asn Lys Lys Thr TyrIle Phe Ala Gly Asp Lys 1 5 10 15 Phe Trp Arg Tyr Asn Glu Val Lys LysLys Met Asp Pro Gly Phe Pro 20 25 30 Lys Leu Ile Ala Asp Ala Trp Asn AlaIle Pro Asp Asn Leu Asp Ala 35 40 45 Val Val Asp Leu Gln Gly Gly Gly HisSer Tyr Phe Phe Lys Gly Ala 50 55 60 Tyr Tyr Leu Lys Leu Glu Asn Gln SerLeu Lys Ser Val Lys Phe Gly 65 70 75 80 Ser Ile Lys Ser Asp Trp Leu GlyCys 85 23 228 PRT Gallus gallus 23 Lys Gly Ile Gln Glu Leu Tyr Glu ValSer Pro Asp Val Glu Pro Gly 1 5 10 15 Pro Gly Pro Gly Pro Gly Pro GlyPro Arg Pro Thr Leu Gly Pro Val 20 25 30 Thr Pro Glu Leu Cys Lys His AspIle Val Phe Asp Gly Val Ala Gln 35 40 45 Ile Arg Gly Glu Ile Phe Phe PheLys Asp Arg Phe Met Trp Arg Thr 50 55 60 Val Asn Pro Arg Gly Lys Pro ThrGly Pro Leu Leu Val Ala Thr Phe 65 70 75 80 Trp Pro Asp Leu Pro Glu LysIle Asp Ala Val Tyr Glu Ser Pro Gln 85 90 95 Asp Glu Lys Ala Val Phe PheAla Gly Asn Glu Tyr Trp Val Tyr Thr 100 105 110 Ala Ser Asn Leu Asp ArgGly Tyr Pro Lys Lys Leu Thr Ser Leu Gly 115 120 125 Leu Pro Pro Asp ValGln Arg Ile Asp Ala Ala Phe Asn Trp Gly Arg 130 135 140 Asn Lys Lys ThrTyr Ile Phe Ser Gly Asp Arg Tyr Trp Lys Tyr Asn 145 150 155 160 Glu GluLys Lys Lys Met Glu Leu Ala Thr Pro Lys Phe Ile Ala Asp 165 170 175 SerTrp Asn Gly Val Pro Asp Asn Leu Asp Ala Val Leu Gly Leu Thr 180 185 190Asp Ser Gly Tyr Thr Tyr Phe Phe Lys Asp Gln Tyr Tyr Leu Gln Met 195 200205 Glu Asp Lys Ser Leu Lys Ile Val Lys Ile Gly Lys Ile Ser Ser Asp 210215 220 Trp Leu Gly Cys 225 24 193 PRT Gallus gallus 24 Leu Cys Lys HisAsp Ile Val Phe Asp Gly Val Ala Gln Ile Arg Gly 1 5 10 15 Glu Ile PhePhe Phe Lys Asp Arg Phe Met Trp Arg Thr Val Asn Pro 20 25 30 Arg Gly LysPro Thr Gly Pro Leu Leu Val Ala Thr Phe Trp Pro Asp 35 40 45 Leu Pro GluLys Ile Asp Ala Val Tyr Glu Ser Pro Gln Asp Glu Lys 50 55 60 Ala Val PhePhe Ala Gly Asn Glu Tyr Trp Val Tyr Thr Ala Ser Asn 65 70 75 80 Leu AspArg Gly Tyr Pro Lys Lys Leu Thr Ser Leu Gly Leu Pro Pro 85 90 95 Asp ValGln Arg Ile Asp Ala Ala Phe Asn Trp Gly Arg Asn Lys Lys 100 105 110 ThrTyr Ile Phe Ser Gly Asp Arg Tyr Trp Lys Tyr Asn Glu Glu Lys 115 120 125Lys Lys Met Glu Leu Ala Thr Pro Lys Phe Ile Ala Asp Ser Trp Asn 130 135140 Gly Val Pro Asp Asn Leu Asp Ala Val Leu Gly Leu Thr Asp Ser Gly 145150 155 160 Tyr Thr Tyr Phe Phe Lys Asp Gln Tyr Tyr Leu Gln Met Glu AspLys 165 170 175 Ser Leu Lys Ile Val Lys Ile Gly Lys Ile Ser Ser Asp TrpLeu Gly 180 185 190 Cys 25 74 PRT Gallus gallus 25 Leu Cys Lys His AspIle Val Phe Asp Gly Val Ala Gln Ile Arg Gly 1 5 10 15 Glu Ile Phe PhePhe Lys Asp Arg Phe Met Trp Arg Thr Val Asn Pro 20 25 30 Arg Gly Lys ProThr Gly Pro Leu Leu Val Ala Thr Phe Trp Pro Asp 35 40 45 Leu Pro Glu LysIle Asp Ala Val Tyr Glu Ser Pro Gln Asp Glu Lys 50 55 60 Ala Val Phe PheAla Gly Asn Glu Tyr Trp 65 70 26 108 PRT Gallus gallus 26 Leu Cys LysHis Asp Ile Val Phe Asp Gly Val Ala Gln Ile Arg Gly 1 5 10 15 Glu IlePhe Phe Phe Lys Asp Arg Phe Met Trp Arg Thr Val Asn Pro 20 25 30 Arg GlyLys Pro Thr Gly Pro Leu Leu Val Ala Thr Phe Trp Pro Asp 35 40 45 Leu ProGlu Lys Ile Asp Ala Val Tyr Glu Ser Pro Gln Asp Glu Lys 50 55 60 Ala ValPhe Phe Ala Gly Asn Glu Tyr Trp Val Tyr Thr Ala Ser Asn 65 70 75 80 LeuAsp Arg Gly Tyr Pro Lys Lys Leu Thr Ser Leu Gly Leu Pro Pro 85 90 95 AspVal Gln Arg Ile Asp Ala Ala Phe Asn Trp Gly 100 105 27 122 PRT Gallusgallus 27 Glu Tyr Trp Val Tyr Thr Ala Ser Asn Leu Asp Arg Gly Tyr ProLys 1 5 10 15 Lys Leu Thr Ser Leu Gly Leu Pro Pro Asp Val Gln Arg IleAsp Ala 20 25 30 Ala Phe Asn Trp Gly Arg Asn Lys Lys Thr Tyr Ile Phe SerGly Asp 35 40 45 Arg Tyr Trp Lys Tyr Asn Glu Glu Lys Lys Lys Met Glu LeuAla Thr 50 55 60 Pro Lys Phe Ile Ala Asp Ser Trp Asn Gly Val Pro Asp AsnLeu Asp 65 70 75 80 Ala Val Leu Gly Leu Thr Asp Ser Gly Tyr Thr Tyr PhePhe Lys Asp 85 90 95 Gln Tyr Tyr Leu Gln Met Glu Asp Lys Ser Leu Lys IleVal Lys Ile 100 105 110 Gly Lys Ile Ser Ser Asp Trp Leu Gly Cys 115 12028 89 PRT Gallus gallus 28 Phe Asn Trp Gly Arg Asn Lys Lys Thr Tyr IlePhe Ser Gly Asp Arg 1 5 10 15 Tyr Trp Lys Tyr Asn Glu Glu Lys Lys LysMet Glu Leu Ala Thr Pro 20 25 30 Lys Phe Ile Ala Asp Ser Trp Asn Gly ValPro Asp Asn Leu Asp Ala 35 40 45 Val Leu Gly Leu Thr Asp Ser Gly Tyr ThrTyr Phe Phe Lys Asp Gln 50 55 60 Tyr Tyr Leu Gln Met Glu Asp Lys Ser LeuLys Ile Val Lys Ile Gly 65 70 75 80 Lys Ile Ser Ser Asp Trp Leu Gly Cys85 29 2123 DNA Gallus gallus CDS (132)..(2123) 29 aattccggca aaagagaaaacggtgcagag agttaagatg tgcagataag caactagtgc 60 actgtgcagc caaagtaactgacagtcagt cagagaaatc ttttaaagag gattgcaaaa 120 atataggcag a atg aag actcac agt gtt ttt ggc ttc ttt ttt aaa gta 170 Met Lys Thr His Ser Val PheGly Phe Phe Phe Lys Val 1 5 10 cta tta atc caa gtg tat ctt ttt aac aaaact tta gct gca ccg tca 218 Leu Leu Ile Gln Val Tyr Leu Phe Asn Lys ThrLeu Ala Ala Pro Ser 15 20 25 cca atc att aag ttc cct gga gac agc act ccaaaa aca gac aaa gag 266 Pro Ile Ile Lys Phe Pro Gly Asp Ser Thr Pro LysThr Asp Lys Glu 30 35 40 45 cta gca gtg caa tac ctg aat aaa tat tat ggatgc cca aaa gac aat 314 Leu Ala Val Gln Tyr Leu Asn Lys Tyr Tyr Gly CysPro Lys Asp Asn 50 55 60 tgc aac tta ttt gta ttg aaa gat act ttg aag aaaatg cag aaa ttt 362 Cys Asn Leu Phe Val Leu Lys Asp Thr Leu Lys Lys MetGln Lys Phe 65 70 75 ttt ggg ctg cct gaa aca gga gat ttg gat caa aac acaatt gag aca 410 Phe Gly Leu Pro Glu Thr Gly Asp Leu Asp Gln Asn Thr IleGlu Thr 80 85 90 atg aag aaa ccc cgc tgt ggt aac ccc gat gtg gcc aat tacaac ttc 458 Met Lys Lys Pro Arg Cys Gly Asn Pro Asp Val Ala Asn Tyr AsnPhe 95 100 105 ttt cca aga aag cca aaa tgg gaa aag aat cat ata aca tacagg att 506 Phe Pro Arg Lys Pro Lys Trp Glu Lys Asn His Ile Thr Tyr ArgIle 110 115 120 125 ata ggc tat acc ccg gat ttg gat cct gag aca gta gatgat gcc ttt 554 Ile Gly Tyr Thr Pro Asp Leu Asp Pro Glu Thr Val Asp AspAla Phe 130 135 140 gcc cga gcc ttt aaa gtc tgg agt gat gtc acg cca ctgaga ttt aac 602 Ala Arg Ala Phe Lys Val Trp Ser Asp Val Thr Pro Leu ArgPhe Asn 145 150 155 cga ata aat gat gga gag gca gac att atg att aat tttggc cga tgg 650 Arg Ile Asn Asp Gly Glu Ala Asp Ile Met Ile Asn Phe GlyArg Trp 160 165 170 gaa cat ggt gat ggc tat cca ttt gat ggc aaa gat ggtctc ctg gct 698 Glu His Gly Asp Gly Tyr Pro Phe Asp Gly Lys Asp Gly LeuLeu Ala 175 180 185 cac gcc ttt gca ccg ggg cca gga att gga gga gac tcccat ttt gat 746 His Ala Phe Ala Pro Gly Pro Gly Ile Gly Gly Asp Ser HisPhe Asp 190 195 200 205 gat gat gaa ctg tgg act ctt gga gaa ggg caa gtggtt aga gta aag 794 Asp Asp Glu Leu Trp Thr Leu Gly Glu Gly Gln Val ValArg Val Lys 210 215 220 tat gga aat gca gat ggt gaa tac tgc aaa ttt cccttc tgg ttc aat 842 Tyr Gly Asn Ala Asp Gly Glu Tyr Cys Lys Phe Pro PheTrp Phe Asn 225 230 235 ggt aag gaa tac aac agc tgc aca gat gca gga cgtaat gat gga ttc 890 Gly Lys Glu Tyr Asn Ser Cys Thr Asp Ala Gly Arg AsnAsp Gly Phe 240 245 250 ctc tgg tgt tcc aca acc aaa gac ttt gat gca gatggc aaa tat ggc 938 Leu Trp Cys Ser Thr Thr Lys Asp Phe Asp Ala Asp GlyLys Tyr Gly 255 260 265 ttt tgt ccc cat gag tca ctt ttt aca atg ggt ggcaat ggt gat gga 986 Phe Cys Pro His Glu Ser Leu Phe Thr Met Gly Gly AsnGly Asp Gly 270 275 280 285 cag ccc tgc aag ttt ccc ttt aaa ttt caa ggccag tcc tat gac cag 1034 Gln Pro Cys Lys Phe Pro Phe Lys Phe Gln Gly GlnSer Tyr Asp Gln 290 295 300 tgt aca aca gaa ggc agg aca gat gga tac agatgg tgt gga acc act 1082 Cys Thr Thr Glu Gly Arg Thr Asp Gly Tyr Arg TrpCys Gly Thr Thr 305 310 315 gaa gac tat gat aga gat aag aaa tac gga ttctgc cca gaa act gcc 1130 Glu Asp Tyr Asp Arg Asp Lys Lys Tyr Gly Phe CysPro Glu Thr Ala 320 325 330 atg tca aca gtt ggt gga aat tca gaa gga gctcct tgt gta ttc ccc 1178 Met Ser Thr Val Gly Gly Asn Ser Glu Gly Ala ProCys Val Phe Pro 335 340 345 ttc atc ttc ctt ggg aat aaa tac gac tcc tgtaca agt gca ggt cgc 1226 Phe Ile Phe Leu Gly Asn Lys Tyr Asp Ser Cys ThrSer Ala Gly Arg 350 355 360 365 aat gat ggc aag ctg tgg tgt gct tct accagc agc tat gat gat gac 1274 Asn Asp Gly Lys Leu Trp Cys Ala Ser Thr SerSer Tyr Asp Asp Asp 370 375 380 cgc aag tgg ggc ttt tgt cca gat caa ggatac agt ctc ttc ttg gtt 1322 Arg Lys Trp Gly Phe Cys Pro Asp Gln Gly TyrSer Leu Phe Leu Val 385 390 395 gct gcc cac gaa ttt ggc cat gcg atg ggatta gag cac tcc gag gac 1370 Ala Ala His Glu Phe Gly His Ala Met Gly LeuGlu His Ser Glu Asp 400 405 410 cca gga gct ctc atg gcc ccg atc tac acctac acc aag aac ttc cgc 1418 Pro Gly Ala Leu Met Ala Pro Ile Tyr Thr TyrThr Lys Asn Phe Arg 415 420 425 ctt tct cag gat gac att aag ggg att caggag cta tat gaa gta tca 1466 Leu Ser Gln Asp Asp Ile Lys Gly Ile Gln GluLeu Tyr Glu Val Ser 430 435 440 445 cct gat gtg gaa cct gga cca ggg ccagga cca ggg cca gga cca cgt 1514 Pro Asp Val Glu Pro Gly Pro Gly Pro GlyPro Gly Pro Gly Pro Arg 450 455 460 cct acc ctt gga cct gtc act cca gagctc tgc aag cac gac att gta 1562 Pro Thr Leu Gly Pro Val Thr Pro Glu LeuCys Lys His Asp Ile Val 465 470 475 ttt gat gga gtt gca caa att aga ggagaa ata ttt ttc ttc aaa gac 1610 Phe Asp Gly Val Ala Gln Ile Arg Gly GluIle Phe Phe Phe Lys Asp 480 485 490 aga ttc atg tgg agg act gta aac cctcga gga aaa ccc aca ggt cct 1658 Arg Phe Met Trp Arg Thr Val Asn Pro ArgGly Lys Pro Thr Gly Pro 495 500 505 ctt ctc gtt gct aca ttc tgg cct gatctg cca gag aaa atc gat gct 1706 Leu Leu Val Ala Thr Phe Trp Pro Asp LeuPro Glu Lys Ile Asp Ala 510 515 520 525 gtc tac gag tcc cct cag gat gagaag gct gta ttt ttt gca gga aat 1754 Val Tyr Glu Ser Pro Gln Asp Glu LysAla Val Phe Phe Ala Gly Asn 530 535 540 gag tac tgg gtt tat aca gcc agcaac ctg gat agg ggc tat cca aag 1802 Glu Tyr Trp Val Tyr Thr Ala Ser AsnLeu Asp Arg Gly Tyr Pro Lys 545 550 555 aaa ctc acc agc ctg gga cta ccccct gat gtg caa cgc att gat gca 1850 Lys Leu Thr Ser Leu Gly Leu Pro ProAsp Val Gln Arg Ile Asp Ala 560 565 570 gcc ttc aac tgg ggc aga aac aagaag aca tat att ttc tct gga gac 1898 Ala Phe Asn Trp Gly Arg Asn Lys LysThr Tyr Ile Phe Ser Gly Asp 575 580 585 aga tac tgg aag tac aat gaa gaaaag aaa aaa atg gag ctt gca acc 1946 Arg Tyr Trp Lys Tyr Asn Glu Glu LysLys Lys Met Glu Leu Ala Thr 590 595 600 605 cca aaa ttc att gcg gat tcttgg aat gga gtt cca gat aac ctc gat 1994 Pro Lys Phe Ile Ala Asp Ser TrpAsn Gly Val Pro Asp Asn Leu Asp 610 615 620 gct gtc ctg ggt ctt act gacagc ggg tac acc tat ttt ttc aaa gac 2042 Ala Val Leu Gly Leu Thr Asp SerGly Tyr Thr Tyr Phe Phe Lys Asp 625 630 635 cag tac tat cta caa atg gaagac aag agt ttg aag att gtt aaa att 2090 Gln Tyr Tyr Leu Gln Met Glu AspLys Ser Leu Lys Ile Val Lys Ile 640 645 650 ggc aag ata agt tct gac tggttg ggt tgc tga 2123 Gly Lys Ile Ser Ser Asp Trp Leu Gly Cys 655 660 30663 PRT Gallus gallus 30 Met Lys Thr His Ser Val Phe Gly Phe Phe Phe LysVal Leu Leu Ile 1 5 10 15 Gln Val Tyr Leu Phe Asn Lys Thr Leu Ala AlaPro Ser Pro Ile Ile 20 25 30 Lys Phe Pro Gly Asp Ser Thr Pro Lys Thr AspLys Glu Leu Ala Val 35 40 45 Gln Tyr Leu Asn Lys Tyr Tyr Gly Cys Pro LysAsp Asn Cys Asn Leu 50 55 60 Phe Val Leu Lys Asp Thr Leu Lys Lys Met GlnLys Phe Phe Gly Leu 65 70 75 80 Pro Glu Thr Gly Asp Leu Asp Gln Asn ThrIle Glu Thr Met Lys Lys 85 90 95 Pro Arg Cys Gly Asn Pro Asp Val Ala AsnTyr Asn Phe Phe Pro Arg 100 105 110 Lys Pro Lys Trp Glu Lys Asn His IleThr Tyr Arg Ile Ile Gly Tyr 115 120 125 Thr Pro Asp Leu Asp Pro Glu ThrVal Asp Asp Ala Phe Ala Arg Ala 130 135 140 Phe Lys Val Trp Ser Asp ValThr Pro Leu Arg Phe Asn Arg Ile Asn 145 150 155 160 Asp Gly Glu Ala AspIle Met Ile Asn Phe Gly Arg Trp Glu His Gly 165 170 175 Asp Gly Tyr ProPhe Asp Gly Lys Asp Gly Leu Leu Ala His Ala Phe 180 185 190 Ala Pro GlyPro Gly Ile Gly Gly Asp Ser His Phe Asp Asp Asp Glu 195 200 205 Leu TrpThr Leu Gly Glu Gly Gln Val Val Arg Val Lys Tyr Gly Asn 210 215 220 AlaAsp Gly Glu Tyr Cys Lys Phe Pro Phe Trp Phe Asn Gly Lys Glu 225 230 235240 Tyr Asn Ser Cys Thr Asp Ala Gly Arg Asn Asp Gly Phe Leu Trp Cys 245250 255 Ser Thr Thr Lys Asp Phe Asp Ala Asp Gly Lys Tyr Gly Phe Cys Pro260 265 270 His Glu Ser Leu Phe Thr Met Gly Gly Asn Gly Asp Gly Gln ProCys 275 280 285 Lys Phe Pro Phe Lys Phe Gln Gly Gln Ser Tyr Asp Gln CysThr Thr 290 295 300 Glu Gly Arg Thr Asp Gly Tyr Arg Trp Cys Gly Thr ThrGlu Asp Tyr 305 310 315 320 Asp Arg Asp Lys Lys Tyr Gly Phe Cys Pro GluThr Ala Met Ser Thr 325 330 335 Val Gly Gly Asn Ser Glu Gly Ala Pro CysVal Phe Pro Phe Ile Phe 340 345 350 Leu Gly Asn Lys Tyr Asp Ser Cys ThrSer Ala Gly Arg Asn Asp Gly 355 360 365 Lys Leu Trp Cys Ala Ser Thr SerSer Tyr Asp Asp Asp Arg Lys Trp 370 375 380 Gly Phe Cys Pro Asp Gln GlyTyr Ser Leu Phe Leu Val Ala Ala His 385 390 395 400 Glu Phe Gly His AlaMet Gly Leu Glu His Ser Glu Asp Pro Gly Ala 405 410 415 Leu Met Ala ProIle Tyr Thr Tyr Thr Lys Asn Phe Arg Leu Ser Gln 420 425 430 Asp Asp IleLys Gly Ile Gln Glu Leu Tyr Glu Val Ser Pro Asp Val 435 440 445 Glu ProGly Pro Gly Pro Gly Pro Gly Pro Gly Pro Arg Pro Thr Leu 450 455 460 GlyPro Val Thr Pro Glu Leu Cys Lys His Asp Ile Val Phe Asp Gly 465 470 475480 Val Ala Gln Ile Arg Gly Glu Ile Phe Phe Phe Lys Asp Arg Phe Met 485490 495 Trp Arg Thr Val Asn Pro Arg Gly Lys Pro Thr Gly Pro Leu Leu Val500 505 510 Ala Thr Phe Trp Pro Asp Leu Pro Glu Lys Ile Asp Ala Val TyrGlu 515 520 525 Ser Pro Gln Asp Glu Lys Ala Val Phe Phe Ala Gly Asn GluTyr Trp 530 535 540 Val Tyr Thr Ala Ser Asn Leu Asp Arg Gly Tyr Pro LysLys Leu Thr 545 550 555 560 Ser Leu Gly Leu Pro Pro Asp Val Gln Arg IleAsp Ala Ala Phe Asn 565 570 575 Trp Gly Arg Asn Lys Lys Thr Tyr Ile PheSer Gly Asp Arg Tyr Trp 580 585 590 Lys Tyr Asn Glu Glu Lys Lys Lys MetGlu Leu Ala Thr Pro Lys Phe 595 600 605 Ile Ala Asp Ser Trp Asn Gly ValPro Asp Asn Leu Asp Ala Val Leu 610 615 620 Gly Leu Thr Asp Ser Gly TyrThr Tyr Phe Phe Lys Asp Gln Tyr Tyr 625 630 635 640 Leu Gln Met Glu AspLys Ser Leu Lys Ile Val Lys Ile Gly Lys Ile 645 650 655 Ser Ser Asp TrpLeu Gly Cys 660 31 21 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide primer 31 attgaattct tctacagttc a 21 32 21 DNAArtificial Sequence Description of Artificial Sequence oligonucleotideprimer 32 atgggatcca ctgcaaattt c 21 33 21 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide primer 33 gccggatccatgaccagtgt a 21 34 21 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide primer 34 gtgggatccc tgaagactat g 21 35 21 DNAArtificial Sequence Description of Artificial Sequence oligonucleotideprimer 35 aggggatcct taaggggatt c 21 36 21 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide primer 36 ctcggatcctctgcaagcac g 21 37 21 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide primer 37 ctcggatcct ctgcaagcac g 21 38 26 DNAArtificial Sequence Description of Artificial Sequence oligonucleotideprimer 38 gcaggatccg agtgctgggt ttatac 26 39 27 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide primer 39 gcagaattcaactgtggcag aaacaag 27 40 26 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide primer 40 gtagaattcc agcactcatttcctgc 26 41 24 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide primer 41 tctgaattct gccacagttg aagg 24 42 21DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide primer 42 attgaattct tctacagttc a 21 43 20 DNAArtificial Sequence Description of Artificial Sequence oligonucleotideprimer 43 gatgaattct actgcaagtt 20 44 21 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide primer 44 cactgaattcatctgcaaac a 21 45 429 PRT Homo sapiens 45 Tyr Cys Lys Phe Pro Phe LeuPhe Asn Gly Lys Glu Tyr Asn Ser Cys 1 5 10 15 Thr Asp Thr Gly Arg SerAsp Gly Phe Leu Trp Cys Ser Thr Thr Tyr 20 25 30 Asn Phe Glu Lys Asp GlyLys Tyr Gly Phe Cys Pro His Glu Ala Leu 35 40 45 Phe Thr Met Gly Gly AsnAla Glu Gly Gln Pro Cys Lys Phe Pro Phe 50 55 60 Arg Phe Gln Gly Thr SerTyr Asp Ser Cys Thr Thr Glu Gly Arg Thr 65 70 75 80 Asp Gly Tyr Arg TrpCys Gly Thr Thr Glu Asp Tyr Asp Arg Asp Lys 85 90 95 Lys Tyr Gly Phe CysPro Glu Thr Ala Met Ser Thr Val Gly Gly Asn 100 105 110 Ser Glu Gly AlaPro Cys Val Phe Pro Phe Thr Phe Leu Gly Asn Lys 115 120 125 Tyr Glu SerCys Thr Ser Ala Gly Arg Ser Asp Gly Lys Met Trp Cys 130 135 140 Ala ThrThr Ala Asn Tyr Asp Asp Asp Arg Lys Trp Gly Phe Cys Pro 145 150 155 160Asp Gln Gly Tyr Ser Leu Phe Leu Val Ala Ala His Glu Phe Gly His 165 170175 Ala Met Gly Leu Glu His Ser Gln Asp Pro Gly Ala Leu Met Ala Pro 180185 190 Ile Tyr Thr Tyr Thr Lys Asn Phe Arg Leu Ser Gln Asp Asp Ile Lys195 200 205 Gly Ile Gln Glu Leu Tyr Gly Ala Ser Pro Asp Ile Asp Leu GlyThr 210 215 220 Gly Pro Thr Pro Thr Leu Gly Pro Val Thr Pro Glu Ile CysLys Gln 225 230 235 240 Asp Ile Val Phe Asp Gly Ile Ala Gln Ile Arg GlyGlu Ile Phe Phe 245 250 255 Phe Lys Asp Arg Phe Ile Trp Arg Thr Val ThrPro Arg Asp Lys Pro 260 265 270 Met Gly Pro Leu Leu Val Ala Thr Phe TrpPro Glu Leu Pro Glu Lys 275 280 285 Ile Asp Ala Val Tyr Glu Ala Pro GlnGlu Glu Lys Ala Val Phe Phe 290 295 300 Ala Gly Asn Glu Tyr Trp Ile TyrSer Ala Ser Thr Leu Glu Arg Gly 305 310 315 320 Tyr Pro Lys Pro Leu ThrSer Leu Gly Leu Pro Pro Asp Val Gln Arg 325 330 335 Val Asp Ala Ala PheAsn Trp Ser Lys Asn Lys Lys Thr Tyr Ile Phe 340 345 350 Ala Gly Asp LysPhe Trp Arg Tyr Asn Glu Val Lys Lys Lys Met Asp 355 360 365 Pro Gly PhePro Lys Leu Ile Ala Asp Ala Trp Asn Ala Ile Pro Asp 370 375 380 Asn LeuAsp Ala Val Val Asp Leu Gln Gly Gly Gly His Ser Tyr Phe 385 390 395 400Phe Lys Gly Ala Tyr Tyr Leu Lys Leu Glu Asn Gln Ser Leu Lys Ser 405 410415 Val Lys Phe Gly Ser Ile Lys Ser Asp Trp Leu Gly Cys 420 425

What is claimed is:
 1. An article of manufacture comprising packagingmaterial and a pharmaceutical agent contained within said packagingmaterial, wherein said pharmaceutical agent is effective for inhibitingangiogenesis in a tissue and wherein said packaging material comprises alabel which indicates that said pharmaceutical agent can be used fortreating conditions by inhibition of angiogenesis and wherein saidpharmaceutical agent comprises an angiogenesis-inhibiting amount of anα_(v)β₃ antagonist, wherein said antagonist is a matrixmetalloproteinase polypeptide that consists of amino acid residuesequence shown in SEQ ID NO 17, 18,19, 20,21, 22, 23, 25, 26, 27 or 28,said polypeptide capable of binding to α_(v)β₃.
 2. The article ofmanufacture of claim 1 wherein said tissue is inflammed and saidcondition is arthritis or rheumatoid arthritis.
 3. The article ofmanufacture of claim 1 wherein said tissue is a solid tumor or solidtumor metastasis.
 4. The article of manufacture of claim 1 wherein saidtissue is retinal tissue and said condition is retinopathy, diabeticretinopathy or macular degeneration.
 5. An α_(v)β₃ antagonist comprisinga matrix metalloproteinase polypeptide that consists of an amino acidresidue sequence shown in SEQ ID NO 17, 18, 19, 20, 21, 22, 23, 25, 26,27 or 28, said polypeptide capable of binding to α_(v)β₃.
 6. Apharmaceutical agent comprising an α_(v)β₃ antagonist according to claim5 in a pharmaceutically acceptable carrier in an amount sufficient toinhibit angiogenesis in a tissue.